Use of isothiocyanates compounds in treating prostatic diseases and skin cancer

ABSTRACT

A method for preventing and treating prostatic diseases and skin cancer using naturally or artificially synthesized isothiocyanates compounds or the derivatives or metabolites thereof.

TECHNICAL FIELD

The invention relates to the application of isothiocyanates or theirderivatives or their metabolites, no matter nature or synthesis, in thetreatment and prevention of benign prostatic hyperplasia (BPH),prostatitis and melanoma.

BACKGROUND

Prostate gland enlargement or benign prostatic hyperplasia (BPH) is acommon disease in aging men. Besides the age, the loss of Phase IIdetoxification enzyme, i.e. glutathione S-transferase (GSTP1) gene, andabnormal of androgen are believed to be involved in the development ofthe disease. The incidence of BPH is quite low in men under age 40,about 40% in men at age 50, and it almost reaches 90% at age 80, nearly100% histochemically in men at age of 90 years old. Among them about 25%of patients need treatments either by surgery or medicine, or both, dueto severe blockage of urinary tract. Obviously, numbers of patients withBPH will significantly increase as a result of the quickening life styleand longer life span. Moreover, recent studies indicate that more BPHoccurs in younger men.

Prostate is consisting of glands and muscles. In those tissues, phase IIdetoxification enzymes are often chronically inactivated with aging byunknown epigenetic reasons. Thus, the ability of cells to defenseinvasion of toxic materials from environment is decreased. As a result,prostate cells are inflammated or extra proliferation of noninflammatedtissue, thereby becoming prostatitis or benign prostatic hyperplasia.Patients with BPH often suffer from urinary difficulty, due to theurethra squeezed by the enlarged prostate. If left untreated andunmonitored, BPH may lead to serious complications, such as frequenturination, urination difficulty, acute urina retention, urinary tractinfection, bladder stone, rectal prolapse, internal hemorrhoids, sexualdysfunction, high blood pressure, heart attack, stroke, urinemia or evenprostate cancer.

The current clinical available pharmaceutical products of BPH can beclassified into three categories: 5-alpha-reductase inhibitors,alpha(1)-receptor antagonists, and natural herbal medicine—Hua Fen.Among them, the Proscar, a 5-alpha reductase inhibitor, manufactured byMerck & Co. Inc, is mainly used for the treatment of BPH. However,clinical studies indicate that Proscar is effective only for patientswith relatively large volume (>40 cm³) of the prostate; otherwise theefficacy is not satisfactory. Since alpha(1)-receptor antagonistsexhibit their activities by relaxation of muscles of bladder neck andtissues surrounding the prostate, it can only relieves symptoms, but notreduce volume of the enlarged prostate.

Prostatitis is another common disease in men of any age, and it is themost common in young and middle-aged men. Prostatitis can be classifiedas two types: acute, bacterial infected inflammation and chronic,non-bacterial infected inflammation. Major symptoms of prostatitisinclude urinating pain, milky urine, urinary fever, frequent urination,pain in lower abdomen; even worse, symptoms like fever or chills.Sometimes symptoms of prostatitis are similar to those of BPH. In thisinvention, we used GENURIN® (Flavoxate Hydrochloride) as positivecontrol for the treatment of prostatitis. This drug is invented andmanufactured by the Lifephama S.r.I, Italy, after its research for manyyears. GENURIN® is indicated to treat bladder and protatic diseases:symptoms like urinary difficulty, urinary urgency, night urine, pain inhaunch bone etc. caused by cystitis and bladder pain, prostatitis,urethritis, bladder urethritis etc. Clinical studies have shown thatthis drug is rapidly absorbed after orally administration, anddistribute into various tissues/organs, and eliminated from urine. Itselectively acts on smooth muscles of genitourinary system, therebyrelieves bladder symptoms caused by irritations.

Incidence of skin cancer is relatively low in Chinese, however it is acommon malignancy in Caucasians. Skin cancer may occur in any part ofthe body, about 80% occur in the skin of the face, head, neck and induceabnormality and danger. UV light, mostly coming from the sun, is a majorfactor to cause skin cancer, thus theoretically, there are nodifferences in incidence of skin cancer between races, skin categories,ages, occupations and locations. Everyone has the possibility to sufferfrom skin cancer. Currently, there are not many available drugs for thetreatment of skin cancer. However patients with skin cancer areincreasing year by year based on related media. A report from WHO inJuly 2006 indicated that there were 60,000 people died, mostly sufferingfrom skin cancer, due to overexposure to sunlight every year. Among them48,000 cases were melanoma, and 12,000 cases were other types of skincancer. Thus, development of new types of pharmaceutical products anddietary supplements to prevent and/or treat skin cancer is highlywarranted.

In summary, prostate diseases are major diseases to affect men health.These diseases will seriously impact our society which is turning intoan aged society. At the same time, more and more patients are sufferingfrom skin cancer. Innovative and effective pharmaceutical products ordietary supplements, food, cosmetics to treat and/or prevent thosedisorders are, therefore, very much needed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention relates to the application of the natural or syntheticisothiocyanates or their derivatives or their metabolites. The inventionfurther relates to the formulations and the preparation methods of theformulations of natural or synthetic isothiocyanates or theirderivatives or their metabolites.

One purpose of this invention is to use the isothiocyanates or theirderivatives or their metabolites to treat and/or prevent the diseasessuch as benign prostatic hyperplasia (BPH), prostatitis and skin canceretc.

The secondary purpose of this invention is to use the isothiocyanates ortheir derivatives or their metabolites as active pharmaceuticalingredients (API) to prepare formulations suitable for treatment and/orprevention of the diseases such as benign prostatic hyperplasia (BPH),prostatitis and skin cancer etc.

The third purpose of this invention is to provide preparation methods offormulations using the isothiocyanates or their derivatives or theirmetabolites as active pharmaceutical ingredients.

This invention relates the natural and synthetic isothiocyanates whichinclude, but not limited to benzyl isothiocyanate (BITC), phenethylisothiocyanate (PEITC), allyl isothiocyanate (AITC) and4-sulfophenylisothiocyanate (SPITC), their chemical structures arelisted below as (1), (2), (3), and (4), respectively:

This invention relates to the derivatives and metabolites ofisothiocyanates. These compounds include, but not limited toisothiocyanate conjugates, including glutathione-, cysteinyl glycine-,cysteinyl-, and N-acetylcysteine-. The preferred conjugate isN-acetylcysteine (NAC) conjugate.

This invention relates to the derivatives and metabolites ofisothiocyanates. These compounds include, but not limited toN-acetylcysteine conjugate of benzyl isothiocyanate (BITC-NAC),N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC),N-acetylcysteine conjugate of allyl isothiocyanate (AITC-NAC), andN-acetylcysteine conjugate of 4-sulfophenylisothiocyanate (SPITC-NAC).Their chemical structures are listed below as (5), (6), (7), and (8),respectively:

The isothiocyanates or their derivatives or their metabolitesdemonstrated in this invention are used for the prevention and treatmentof diseases of the prostate and skin cancer. The compounds shown thegood efficacy are phenethyl isothiocyanate (PEITC), benzylisothiocyanate (BITC) and their N-acetylcysteine conjugates. Thecompounds shown the better efficacy are phenethyl isothiocyanate (PEITC)and N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC).The compound shown the best efficacy is phenethyl isothiocyanate(PEITC).

The isothiocyanates or their derivatives or their metabolites involvedin this invention are used for the treatment and/or prevention ofdiseases of the prostate and skin cancer. The preferred diseases of theprostate are benign prostatic hyperplasia (BPH) and prostatitis,particularly the benign prostatic hyperplasia.

The isothiocyanates or their derivatives or their metabolites involvedin this invention include, but not limited to phenethyl isothiocyanate(PEITC), benzyl isothiocyanate (BITC), allyl isothiocyanate (AITC),4-sulfophenylisothiocyanate (SPITC) and N-acetylcysteine conjugate ofphenethyl isothiocyanate (PEITC-NAC). All of those compounds are able toeffectively induce expressions of phase II detoxification enzyme, i.e.glutathione S-transferase (GSTP1) gene, enable prostate cells toeliminate invasions of toxic materials. Therefore, those compounds areable to effectively treat and/or prevent inflammations and other relateddiseases of the prostate and other tissues/organs.

The isothiocyanates or their derivatives or their metabolites involvedin this invention include, but not limited to phenethyl isothiocyanate(PEITC), benzyl isothiocyanate (BITC), allyl isothiocyanate (AITC) andN-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC).Those compounds effectively repress expressions of the androgen receptor(AR), transcription factor Sp1, an upstream gene of AR, and prostatespecific antigen (PSA), a downstream gene of the AR.

Compositions containing one or more active pharmaceutical ingredients(API) described in this invention can be used as pharmaceuticalproducts, food, dietary supplements, or cosmetics.

Compositions described in this invention contain one or more of theisothiocyanates or their derivatives or their metabolites as APItogether with other ingredients listed below:

(a) 0.5-50 portion (w/w) of API, which includes isothiocyanates and/ortheir derivatives, their metabolites. The isothiocyanates include, butnot limited to phenethyl isothiocyanate (PEITC), benzyl isothiocyanate(BITC), allyl isothiocyanate (AITC), 4-sulfophenylisothiocyanate(SPITC). The derivatives herein include, but not limited toisothiocyanate conjugates of N-acetylcysteine. The preferred compoundsare N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC),N-acetylcysteine conjugate of benzyl isothiocyanate (BITC-NAC),N-acetylcysteine conjugate of allyl isothiocyanate (AITC-NAC), andN-acetylcysteine conjugate of 4-sulfophenylisothiocyanate (SPITC-NAC).

(b) 0 to 1000 portion (w/w) of API pharmaceutical carrier, thepharmaceutical carrier include:

(b1) 0 to 1000 portion (w/w) of pharmaceutical surfactants orsolubilizing agents, the described surfactants were selected from:polyoxyethylene lauryl ether (preferentially polyoxyethylene 23 laurylether), polyoxyethylene glycol stearate (preferentially, Polyoxyethylene(40) monostearate), Vitamin E polyethylene glycol succinate,polyoxyethylene castor oil (preferentially, polyoxyethylene (35) castoroil), polyoxyethylene hydrogenated castor oil (preferentially,Polyoxyethylene (40) hydrogenated castor oil), poloxamer(preferentially, poloxamer-F127, poloxamer F-68), polysorbates (Tween80) or their combination. The described solubilizing agents include:polyvinylpyrrolidone K17, K25, K30, K90, polyethylene glycol 400, 4000,and 6000 or their combination. Preferentially, the describedsurfactants, and solubilizing agents are polyoxyethylene (40)monostearate, polyoxyethylene 23 lauryl ether, Vitamin E polyethyleneglycol succinate, polyoxyethylene (40) hydrogenated castor oil or theircombination, particularly, polyoxyethylene (40) monostearate.

(b2) 0 to 500 portions (w/w) of oil components, were selected from fattyacids or triglyceride, monoglyceride or diglyceride, which include, butnot limited to soybean oil, corn oil, peanut oil, stearic acid, palmiticacid, palm oil, sunflower oil, olive oil, coconut oil, sesame oil,cottonseed oil, canola oil, oleic acid, linoleic acid, medium-chaintriglycerides, glyceryl monooctadecanoate, glyceryl monoacetate,glyceryl diacetate, glyceryl triacetate, or one or more than onecombination. Preferentially, the described oil components were selectedfrom: medium-chain triglycerides, triglyceride, soybean oil, corn oil,cottonseed oil, stearic acid, oleic acid, particularly: medium-chain(i.e. C8 to C12) triglycerides, soybean oil, corn oil, and the best oneis medium-chain triglycerides.

(b3) 0 to 25 portions (w/w) of antioxidants are selected from:water-soluble antioxidants, fat-soluble antioxidant, such as vitamin C,vitamin C palmitate, propyl gallate, vitamin E (tocopherol),tert-butylated-p-hydroxyanisole, 2,6-di-tert-butyl-p-methylphenol, orcombination of one or more than one of the above components.Preferentially, those antioxidants are vitamin C, vitamin C palmitate,propyl gallate, tert-butylated-p-hydroxyanisole, and the best choice isvitamin C palmitate.

(b4) is one or more than one of combination of (b1), (b2), and (b3).

To further prepare other suitable dosage forms, the following excipientsmay be added into the pharmaceutical products and dietary supplementsdescribed in this invention:

(a) Adsorbents and diluents: the adsorbents and diluents are selectedfrom α-lactose(monohydrate), anhydrous lactose, β-cyclodextrin,hydroxypropyl-β-cyclodextrin, microcrystalline cellulose (MCC, PH101,PH102, KG series), microcrystalline cellulose pills, lactose starchpills, calcium carbonate, calcium hydrocarbonate, modified starch,sucrose octaacetate, sodium carboxymethyl starch, hydroxypropylcellulose, stearic acid, mannitol, sorbitol, sorbic acid, sodiumcarboxymethyl cellulose, porous starch, colloidal silicon dioxide, orthe combination of one or more than one of components described above.Preferentially, adsorbents and diluents are stearic acid, sorbitol,microcrystalline cellulose (KG series), mannitol 300 DC, β-cyclodextrin,colloidal silicon dioxide, α-lactose(monohydrate). The better ones areβ-cyclodextrin, stearic acid and the best one is β-cyclodextrin. Thedescribed amount of adsorbents and diluents are between 0.5 and 1500portions (w/w), and the better ranges are between 2.0 and 500 portions(w/w).

(b) Lubricants: The lubricants are selected from magnesium stearate,lauryl sodium sulfate, polyethylene glycol, colloidal silicon dioxideand talc. Preferentially, lubricants are magnesium stearate,polyethylene glycol and colloidal silicon dioxide. The best one ispolyethylene glycol, and the ratio is between 0 and 30 portions (w/w).

(c) Binders: The binders described in this invention are selected frompolyvinylpyrrolidone (PVP), hydroxypropyl cellulose, hydroxypropylmethylcellulose, ethyl cellulose (200 mesh), polyethylene glycol (PEG).Preferentially, binders are selected from hydroxypropyl cellulose EF,PEG 6000, hydroxypropylmethyl cellulose E15. The better choices arehydroxypropylmehtyl cellulose E15, PEG 6000, and the best one is PEG6000. The amount of the binder usually ranges between 0 and 100 portions(w/w).

(d) Disintegrating agents: The disintegrating agents described in thisinvention are selected from: porous starch, sodium carboxymethylcellulose, sodium carboxymethyl starch, low-substituted hydroxypropylcellulose, microcrystalline cellulose. The preferential selections ofdisintegrating agents are microcrystalline cellulose PH101,low-substituted hydroxypropyl cellulose and sodium carboxymethylcellulose, and the best one is microcrystalline cellulose PH101. Theamount of disintegrating agents is usually between 0 and 100 portions(w/w).

(e) Coating materials: The coating materials described in this inventionare selected from: hydroxypropylmethyl cellulose, hydroxypropylcellulose, methyl cellulose, ethyl cellulose,dimethylaminoethylmethylacrylate-neutral methylacrylate polymer, PEG,titanium dioxide, iron oxide red, and potassium sorbate. Among them, thedescribed coating materials are preferentially selected fromhydroxypropylmethyl cellulose E15, hydroxypropyl cellulose EF,dimethylaminoethylmethylacrylate-neutral methylacrylate polymer, PEG400, PEG 4000, and ethyl cellulose (200 mesh), and more preferentiallyselected from hydroxypropylmethyl cellulose E15, PEG 4000, and ethylcellulose. The amount of the coating materials is usually from 0.5 to 20portions (w/w).

In an preferred pharmaceutical product or dietary supplement, the amountby weight are APIs from 1 to 25 portions (w/w), surfactants from 5 to200 portions (w/w), oil components from 0.5 to 100 portions (w/w),antioxidants from 1 to 20 portions (w/w), and other excipients from 0.5to 1500 portions (w/w).

The compositions of the pharmaceutical products and dietary supplementsdescribed in this invention can be manufactured, at least, into tablet,capsule, pill, powder for injection, injection, lyophilized powder,ointment, suppository, cream, film, emulsion, spray or implant.

The formulations of the pharmaceutical products and dietary supplementsdescribed in this invention can be administered orally, intravenously,muscularly, subcutaneously, intracavitaryly, sublingually, anally, ortopically.

The formulations of the pharmaceutical products and dietary supplementsdescribed in this invention can be used to treat and/or preventprostatic diseases and skin cancer. The described prostatic diseases arepreferentially referred to benign prostatic hyperplasia and prostatitis,particularly the benign prostatic hyperplasia.

Among the composition of the pharmaceutical products and dietarysupplements described in this invention, the preferred ones haveadvantages of high dissolution rate, better stability, low doses, andfewer side effects.

Among the composition of the pharmaceutical products and dietarysupplements described in this invention, the preferred ones containphenethyl isothiocyanate (PEITC) as the active pharmaceutical ingredient(API), which can effectively inhibit and reduce the abnormal hyperplasiaof the prostate tissue.

Among the composition of the pharmaceutical products and dietarysupplements described in this invention, the preferred ones containphenethyl isothiocyanate (PEITC) as the active pharmaceutical ingredient(API), which can effectively prevent and/or treat the inflammation ofthe prostate tissue.

Among the composition of the pharmaceutical products and dietarysupplements described in this invention, the preferred ones containphenethyl isothiocyanate (PEITC) as the active pharmaceutical ingredient(API), which can effectively inhibit the proliferation of skin cancercells.

The dosage ranges of the composition containing one or more than one ofisothiocyanates described in this invention for the treatment of benignprostatic hyperplasia and prostatitis are between 0.1 and 20 mg/kg,preferentially between 1 and 4 mg/kg. We demonstrated in this inventionthat among three different doses, i.e. 1, 2, and 4 mg/kg used in ourexperiments, the lowest dose of 1 mg/kg has already shown therapeuticactivities against benign prostatic hyperplasia and prostatitis.

The composition of the pharmaceutical products and dietary supplementsdescribed in this invention, particularly those containing phenethylisothiocyanate (PEITC) as API, can be used for the treatment of thediseases alone, or in combination with other therapies, such as surgery,one or more than one of the Western medicine or the Traditional ChineseMedicine, radiation, gene therapy and biologics etc.

In comparison with existing technology, the advantages of this inventionare:

This invention demonstrates, for the first time, that isothiocyanates,including PEITC etc., effectively treat and/or prevent benign prostatichyperplasia and prostatitis.

This invention further demonstrates that other isothiocyanates aresimilar to PEITC and are able to induce expression of phase IIdetoxification enzyme, i.e. glutathione S-transferase gene. All of thoseisothiocyanates are capable of increasing the ability of the prostaticcells to eliminate the toxic materials, thereby effectively treatingand/or preventing prostate diseases and inflammations of othertissues/organs.

This invention demonstrates in a castrated rat model, for the firsttime, that isothiocyanates as exampled by PEITC are effectively againstBPH.

This invention demonstrates in a rat model, for the first time, thatisothiocyanates as exampled by PEITC can effectively treat and preventprostatitis, particularly non-bacterial prostatitis.

This invention demonstrates for the first time that isothiocyanatessignificantly repress expressions of the androgen receptor (AR), Sp1, aupstream of AR gene, and prostate specific antigen (PSA), a downstreamof AR gene in human prostate cancer cells.

This invention demonstrates for the first time that isothiocyanates asexampled by PEITC effectively inhibit the growth of B16 melanoma cells(skin cancer cell line).

This invention further provides, for the first time, formulationscontaining API of one or more of natural or synthetic isothiocyanatesincluding PEITC etc.

This invention demonstrates that composition containing API of theisothiocyanates as exampled by PEITC is effective against BPH andprostatitis at low doses.

This invention demonstrates that composition of the pharmaceuticalproducts and dietary supplements containing API of the isothiocyanatesincluding PEITC etc. for the treatment of BPH and prostatitis has betterdissolution rates, proved efficacy, minor side effects. They areeffective at low doses, and are stable.

This invention provides a method of treating diseases of prostate. Thedescribed diseases of the prostate include, but not limited to BPH andprostatitis. The amount or dose of isothiocyanates or their derivativesor their metabolites in these applications is between 0.1 mg/kg and 20mg/kg.

In one of particular example described in this invention, the dose ofAPI at 1 mg/kg achieved good inhibitory therapeutic effect on BPH andprostatitis.

This invention further provides a method to repress expression of PhaseII detoxification enzyme gene. The method includes following procedure:to expose prostate cells to one or more of isothiocyanates or theirderivatives or their metabolites for certain period of time, and induceexpression of the GSTP1 gene.

This invention also provides a method to treat skin cancer by using oneor more of isothiocyanates or their derivatives or their metabolites towhom needed.

BRIEF DESCRIPTION OF FIGURES

FIG. 1, Induction of phase II detoxification enzyme GSTP1 by variousisothiocyanates in the human prostate cancer cell line LNCaP. PEITC:phenethyl isothiocyanate, BITC: benzyl isothiocyanate: AITC:allylisothiocyanate, SPITC: 4-sulfophenylisothiocyanate, and PEITC-NAC:N-acetylcysteine conjugate of phenethylisothiocyanate.

FIG. 2, Effect of phenethyl isothiocyanate (PEITC) on transcriptionfactor, Sp1.

FIG. 3, Inhibition of phenethyl isothiocyanate (PEITC) on binding of Sp1to the promoter DNA of androgen receptor.

FIG. 4, Inhibitory effects of various isothiocyanates on the expressionsof AR, AR upstream gene, Sp1, and AR downstream gene, prostate specificantigen (PSA) of the human prostate cancer cell line LNCaP. PEITC:phenethyl isothiocyanate, BITC: benzyl isothiocyanate, AITC: allylisothiocyanate, SPITC: 4-sulfophenylisothiocyanate, and PEITC-NAC:N-acetylcysteine conjugate of phenethyl isothiocyanate.

FIG. 5, A typical histological slide section of normal prostate tissues(amplified by 20 times).

FIG. 6.1, A typical slide section of increasing density of prostaticacinars of benign prostatic hyperplasia (BPH) tissues of negativecontrol (amplified by 20 times). This slide showed that the density ofprostatic acinars was significantly increased as compared with normalprostatic gland.

FIG. 6.2, A typical slide section of enlarging lumen of prostaticacinars of benign prostatic hyperplasia (BPH) tissues of negativecontrol (amplified by 20 times). The slide showed that the lumen ofprostatic gland and secretary materials in the lumen of BPH weresignificantly increased as compared with normal prostatic gland.

FIG. 6.3, Effects of Proscar on the lumen of prostatic gland. This is atypical slide of benign prostatic hyperplasia from animal treated withpositive control drug, Proscar (amplified by 20 times). The slide showedthat the lumen of prostatic gland and secretary materials in the lumenof BPH were increased as compared with normal prostatic gland. At thesame time the slide also showed that they were reduced as compared withuntreated benign prostatic hyperplasia tissues of negative control.

FIG. 6.4, Effects of Proscar on the density of prostatic acinars. Thisis a typical slide of benign prostatic hyperplasia from animal treatedwith positive control drug, Proscar (amplified by 20 times). This slideshowed that density of prostatic acinars was increased as compared withnormal prostatic gland, but the density was reduced as compared withuntreated BPH tissues of negative control.

FIG. 6.5, Effects of PEITC (2 mg/kg) on BPH (amplified by 20 times).This slide showed that the lumen, secretary materials in the lumen, andthe density of prostatic acinars in BPH animals treated with 2 mg/kgPEITC were increased as compared with normal prostatic gland. But theywere reduced as compared with untreated BPH tissues of negative control.

FIG. 7.1, Histological section of the prostatitis from an untreatedprostatitis animal of negative control (amplified by 20 times). Thisslide showed congestion, edema, leukocyte infiltration and bleeding inthe inflammatory prostatic gland.

FIG. 7.2, Histological section of the lymphocyte infiltration from ananimal treated with Genurin (amplified by 20 times). This slide showedlymphocyte infiltration, fibrosis, and congestion.

FIG. 7.3, Histological section of the prostatitis from an animal treatedwith PEITC at dose of 4 mg/kg (amplified by 20 times). The slide showedthat the prostatic tissues have edema, slight congestion and fibroidinfiltration, but the histopathological scores were less severe thanthat of prostatic tissues from untreated prostatitis animals of negativecontrol.

FIG. 7.4, Histological section of the prostatitis from an animal treatedwith PEITC at dose of 2 mg/kg (amplified by 20 times). The slide showedthat the prostatic tissues have congestion, edema and fibroidinfiltration.

FIG. 7.5, Histological section of the prostatitis from an animal treatedwith PEITC at dose of 1 mg/kg (amplified by 20 times). The slide showedthat the prostatic tissues have congestion, edema and fibroidinfiltration, leukocyte infiltration but they were less severe than thatof prostatic tissues from untreated prostatitis animals of negativecontrol.

FIG. 8, Comparison of cell growth inhibition among differentisothiocyanates in the human prostate cancer cell line LNCaP. PEITC:phenethyl isothiocyanate, BITC: benzyl isothiocyanate: AITC: allylisothiocyanate, SPITC: 4-sulfophenylisothiocyanate.

FIG. 9, Effects of phenethyl isothiocyanate (PEITC) on expression ofendogenous androgen receptor (AR) and AR downstream gene-prostatespecific antigen (PSA).

FIG. 10, Effects of phenethyl isothiocyanate (PEITC) on growth curve ofB16 (mouse melanoma cell line)

FIG. 11, Effects of phenethyl isothiocyanate (PEITC) on volumeenlargement of mouse melanoma cell line by gavage.

EXAMPLES Example 1 Preparation of Phenethyl Isothiocyanate (PEITC)

Instrument and Reagents:

¹H-NMR: Brucker AV-300, TMS was used as an internal standard, and CDCl₃as solvent otherwise as indicated; Mass Spectrometer (MS): NicoletFTMS-2000; Element analyzer: Elementar Vario EL III.

Thin layer Chromatography (TLC): Silicon GF₂₅₄ (Qingdao Ocean ChemicalPlant, Qingdao, China) was used to prepare TLC plates. All reagents witheither chemical grade or analytical grade were directly used withoutfurther treatment.

Example 1.1 Extraction of Phenethyl Isothiocyanate (PEITC) from NaturalPlants

a) Watercress was chopped and dipped in water for a few days under roomtemperature. The phenethyl isothiocyanate (PEITC) was produced byendogenous enzyme of the watercress during this procedure, and wasextracted with water insoluble solvent such as N-hexane. The solvent wasthen removed by vacuum and the PEITC obtained with relative high yieldand purity.

b) Phenethyl isothiocyanate (PEITC) could be also prepared from gardencress using the same method as above. After dipping the root of GrassOleaceae in water, use water vapore distillation to obtain phenethylisothiocyanate.

Example 1-2 Synthetic Preparation of Phenethyl Isothiocyanate (PEITC)

Fifteen ml of CH₂Cl₂ and 3 ml (40 mmol) of thiosulfate phosgene wereadded into a 50 ml round bottomed-flask, stirred and cooled to 0° C.Equivalent amount of triethylamine (4.04 g, 40 mmol) was then slowlyadded using a constant pressure drop liquid funnel (heat would bereleased, and the temperature should be controlled no higher than 15°C.). When it was finished, the reaction mixture was allowed to react foradditional 5-6 hrs at room temperature. After the reaction was completed(no triethylamine was present in the reaction mixture as monitored byTLC), 10 ml of H₂O was added to terminate the reaction. Additional 5 mlof CH₂Cl₂ was then added, and the organic phase was separated by afunnel, washed twice with water (15 ml×2), dried with anhydrous sodiumsulfate, filtered, and condensed to dry. The residue was purified andeluted using petroleum ether (boiling point was between 60 and 90° C.)in a silicon column. After condensation and vacuum distillation, 4.9 gcolorless and oily liquid of PEITC was obtained. The yield wasapproximately 75% and its chemical structure was confirmed by using NMRand MS as described by Katritzky and Victor Gil., et al (Alan R.Katritzky et al. 1979; Victor Gil et al, 1980).

Data of Structure Characterization:

¹H-NMR δ: 7.26-7.24 (m, 3H, Ph-H), 7.12 (d, J=8.5 Hz, 2H, Ph-H), 3.94(t, J=7.0 Hz, 2H, CH₂), 2.81 (t, J=7.0 Hz, 2H, CH₂); ESI-MS:164.1[M+H]⁺, C₉H₉NS (163.24); Anal. Calcd for C₉H₉NS:C66.22, H5.56,N8.58. Found: C66.30, H5.42, N8.34; Molecular weight: 163.24.

Example 2 Effects of Various Isothiocyanate on Expression of GSTP1Protein

Materials and Methods:

Reagents:

PEITC was synthesized by Wuxi JC Pharmaceutical Technology Co., Ltd. andits chemical structure was confirmed by NMR, UV, IR and MS and thecontent and purity was analyzed by HPLC (>99%). Other isothiocyanates,benzyl isothiocyanate (BITC), allyl isothiocyanate (AITC), and4-sulfophenylisothiocyanate (SPITC) were purchased from Sigma-Aldrichcompany (USA). N-acetylcysteine conjugate of phenethyl isothiocyanate(PEITC-NAC) was purchased from LKT Company (USA). The other chemicalswere purchased from Sigma (St. Louis, Mo.) otherwise as indicated.Reagents for protein electrophoresis and nitrocelluse membrane werepurchased from Bio-Rad Company (USA). The ELC Western detection kit andfilms were purchased from GE (USA). Antibodies against phase IIdetoxification enzyme GSTPI and β-actin, as well as the second antibodywere purchased from BD and Santa Cruz Biotechnology, Inc. (USA),respectively.

Cell Culture:

The human prostate cancer cell lines LNCaP and PC-3 were purchased fromthe American Type Culture Collection (Rockville, Md.) and weremaintained in RPMI-1640 (GIBCO, Gaithersburg, Md.) with 10% fetal bovineserum (FBS), penicillin—streptomycin and incubated at 5% CO₂ and 37° C.

Western blot to detect GSTP1 protein (FIG. 1): LNCaP cells atexponential growth phase were exposed to indicated concentrations ofPEITC, BITC, AITC, SPITC, and PEITC-NAC for 24 hrs. The cells wereharvested, washed and total cellular proteins were extracted asdescribed previously (Wang, L. G., L. Ossowski, et al. Overexpressedandrogen receptor linked to p21WAF1 silencing may be responsible forandrogen independence and resistance to apoptosis of a prostate cancercell line. Cancer Res 61(20): 7544-51, 2001). Fifty μg cellular extractswere separated on a SDS-PAGE, electro-transferred to nitrocellulosefilters, and immunoblotted initially with antibodies against GSTP1. Thesame membranes were stripped and re-probed with β-actin for loadingcontrol. The result was recorded by ECL films.

Results and Discussion:

Using Western blot, we explored possible mechanisms by whichisothiocyanates are effective against benign prostatic hyperplasia andprostatitis. As shown in FIG. 1, all isothiocyanates tested in thisexample under the same experimental conditions showed significantinduction of GSTP1 expression. As a phase II detoxification enzyme,GSTP1 is able to effective neutralize endogenous and exogenous harmfulagents including inflammatory components and carcinogens. It has beendemonstrated that the GSTP1 gene was gradually shut off during thedevelopment of prostate diseases including prostatitis, benign prostatichyperplasia and prostate cancer due to hypermethylation of CpG island inthe promoter region of the GSTP1 gene. In this example, we clearlyshowed that PEITC and all other tested isothiocyanates which haveisothiocyano-function group, significantly restored GSTP1 geneexpression (FIG. 1). Thus, restoration of GSTP1 expression may be amajor mechanism by which tested isothiocyanates effectively treat andprevent benign prostatic hyperplasia and prostatitis due to theirisothiocyano-function group. Our study also demonstrates thatisothiocyanates with isothiocyano-function group include4-sulfophenylisothiocyanate (SPITC) and the metabolite (PEITC-NAC) ofPEITC are effective agents for the prevention and treatment of prostatediseases.

Example 3 Inhibitory Effects of PEITC on Sp1 Transcription Factor

Materials and Methods

Reagents:

PEITC synthesized as Example 1-2 was dissolved in DMSO. The Sp1-luc andmtSp1-luc contain three tandem repeats of consensus Sp1 sites drivingthe luciferase gene and its mutant. They were used to evaluate theeffects of PEITC on Sp1 expression. Effectene transfection reagent andluciferase assay kit were acquired from Qiagen (Valencia, Calif., USA)and Promega (Madison, Wis., USA). Reagents for protein electrophoresiswere purchased from Bio-Rad company (USA). The other chemicals werepurchased from Sigma (St. Louis, Mo.) otherwise as indicated.

Cell Culture:

The human prostate cancer cell line LNCaP AD was purchased from theAmerican Type Culture Collection. LNCaP AI cell line was derived fromLNCaP cells using a method described previously by Gao et al. (Gao, M.,Ossowski, L., and Ferrari, A. C. Activation of Rb and decline inandrogen receptor protein precede retinoic acid-induced apoptosis inandrogen-dependent LNCaP cells and their androgen-independentderivative. J Cell Physiol, 179: 336-346, 1999). The AD and AI cellswere maintained in RPMI-1640 with 10% fetal bovine serum (FBS) andcharcoal stripped fetal bovine serum, penicillin-streptomycin forincubation at 5% CO₂ and 37° C. respectively.

Gene Transfection:

LNCaP AD or LNCaP AI cells grown exponentially were seeded into 60 mmdishes at a density of 10⁵ per ml. After incubation for 24 hrs at 5% CO₂and 37° C., the cells were transfected with 1 μg/dish of SP1-luciferasegene (Sp1-luc), or its mutant mtSp1-luc using Effectene (Qiagen,Valencia, Calif.) as the transfection reagent. Twenty-four hrs aftertransfection, the cells were exposed for an additional 24 hrs to variousconcentrations of JC-5411(PEITC). The cells were collected, washed,lysed, and the lysates were used for luciferase activity assay usingPromega luciferase assay system.

Mobility Gel Shift Assay (EMSA):

EMSA was performed according to modern technology. Briefly, AD cellsgrown exponentially were exposed to PEITC for 24 hrs. The extraction ofnuclear proteins was performed as described previously (Wang, L. G.,Liu, X. M., Kreis, W., and Budman, D. R. Down-regulation ofprostate-specific antigen expression by finasteride through inhibitionof complex formation between androgen receptor and steroidreceptor-binding consensus in the promoter of the PSA gene in LNCaPcells. Cancer Res, 57: 714-719, 1997). Five μg of nuclear proteins wasreacted for 30 minutes at room temperature with the [³²P]-labeled Sp1oligonucleotide or their corresponding mutants oligonucleotide probe inbinding buffer containing 1 g dIdC to increase the specificity of thereaction. The reaction mixtures were then subjected to electrophoresisin 8% native polyacrylamide gel(PAGE). The binding complexes werevisualized by exposing the dried gel to X-ray film.

Results and Discussion:

In order to examine whether PEITC mediated inhibition of transcriptionfactor SP1, the effects of PEITC on Sp1 specific luciferase transfectiongene was investigated in which an Sp1 specific activation agent,trichostatin A (TSA) and Sp1 specific inhibitor, Mithramycin were usedas controls to ensure the reliability of the test system. As shown inFIG. 2, treatment of SP1-luc transfected AD cells with PEITC for 24 hrs,the luciferase activity of transfected gene was significantly decreasedwhich was similar to the positive control drug, Mithramycin. Theinhibitory activity of PEITC on Sp1 expression was also found to beconcentration dependent. In contrast, TSA significantly stimulated theluciferase activity. But no activity was observed when the mSp1-luc wasused. This result demonstrated that PEITC is the transcription factorSp1 inhibitor.

To further explore possible mechanisms of PEITC inhibiting transcriptionfactor Sp1, mobility gel shift assay (EMSA) was performed to examineeffects of PEITC on Sp1-DNA binding. Sp1 is a transcription factor andits regulatory activity is through its binding to the SP1 binding siteof the target gene promotor. EMSA is a common method to study the role,As shown in FIG. 3, after the treatment with PEITC, the binding activityto Sp1 specific oligonucleotide of nuclear proteins was significantlyreduced in a concentration-dependent manner. This result not onlyindicates that PEITC is an Sp1 inhibitor, but also suggests that effectof PEITC on Sp1 downstream targets, such as SP1 specific luciferasetransfection gene, is through its decreased Sp1-DNA binding complexformation.

Example 4 Effects of Various Isothiocyanates on AR, Sp1 and PSA Proteins

Materials and Methods:

Reagents:

Isothiocyanates were the same as Example 2. Other chemicals werepurchased from Sigma (St. Louis, Mo.) otherwise as indicated. Reagentsfor protein electrophoresis and nitrocelluse membrane were purchasedfrom Bio-Rad company (USA). The ELC Western detection kit and films werepurchased from GE (USA). Antibodies against the AR, Sp1, PSA and β-actinetc. were purchased from BD Biosciences (USA) and Santa CruzBiotechnology, Inc. (USA).

Cell Culture:

The same procedures were used as described in Example 2.

Western blot to detect AR, SP1, PSA:

The human prostate cancer cell line LNCaP at exponential growth phasewere exposed to PEITC, BITC, AITC, SPITC and PEITC-NAC for 24 hrs (shownin FIG. 4). The cells were harvested, washed and total cellular proteinswere extracted as described previously (as Example 2). Fifty μg proteinswere then subjected to electrophoresis in SDS-PAGE, electro-transferredto nitrocellulose filters, and immunoblotted initially with antibodiesagainst AR, Sp1, and PSA. The same membranes were stripped and re-probedwith β-actin for loading control. The result was recorded by ECL films.

Results and Discussion:

To further elucidate the effect of PEITC on SP1, other possiblemolecular mechanisms of isothiocyanates against growth of benignprostatic hyperplasia, prostatitis, prostate cancer was investigated byWestern blot. The results showed that some isothiocyanates, particularlyPEITC and BITC significantly inhibited expression of the AR, upstreamgene of AR:Sp1 and downstream gene of AR:PSA (Shown in FIG. 4). WhilePEITC-NAC showed similar effects on those gene expressions, AITC onlyshowed minimal activities, and no effect was achieved when the cellswere treated with SPITC under the same experimental conditions. Theresults were in accordance with the inhibitory effect of theseisothiocyanates on LNCaP cancer cells (Example 8). It is indicated thatthe inhibitory activity of these compounds on AR expression may be amolecular mechanism involved in selectively inhibiting proliferation ofBPH and prostate cancer.

Based on the molecular mechanism study of the inhibitory effect of theisothiocyanates on BPH, prostatitis and prostate cancer, the in vitroand in vivo pharmacodynamics of the isothiocyanates were furtherstudied.

Example 5 Therapeutic Effects of PEITC on BPH: Test One

Materials and Methods

Reagents:

PEITC was the same as Example 2. Testosterone propionate, estradiol,ketamine, and penicillin were purchased from Wuxi Shanhe Health DrugChain Limited Participation Group.

Animals:

Adult pathogen-free SD rats, male, body weight ranging from 150 to 200grams, were purchased from the Animal Center of Nanjing MedicalUniversity. The animals were housed five per cage and fed ad libitum,fresh tap water and commercial rodent pellets. Animal rooms werecontrolled at 25±2° C. and a 12 hrs light/dark cycle.

Disease Model and Drug Treatment:

Sixty male SD rats were randomly divided into 6 groups, ten each. Theanimals were anesthetized using ketamine and castrated by removingtestis under sterile conditions. After the surgery, the penicillin wasinjected to prevent the animals from infection. One week later, thetestosterone propionate was injected subcutaneously at dose of 1 mg/ratfor a month, once a day. Group 1 was served as a negative control group,and the animals of group 1 were given saline orally without any activepharmaceutical ingredient for a month beginning on the same day thetestosterone propionate was injected. Group 2 was served as positivecontrol group in which animals were given estradiol subcutaneously at adose of 0.1 mg/rat for a month beginning on the same day thetestosterone propionate was injected. Groups from 3 to 6 was served asdrug treatment groups in which animals were given tested articles on thesame day the testosterone propionate was injected. The animals in groupsfrom 3 to 6 were given PEITC composition (oil base and aqueous base) fora month, once a day, by gavage at doses of 4 mg/kg (API of theFormulation of Example 13-3), 1 mg/kg (API of the Formulation of Example13-3), 4 mg/kg (API of the Formulation of Example 13-11) and 1 mg/kg(API of the Formulation of Example 13-11), respectively.

Twenty-four hrs after the last dosage, the animals were euthanized andweighed. Volume and weight of prostate were measured. The averages ofvolume/weight and organ coefficient (average weight of prostate/averageof body weight) were calculated, and statistical analysis performed (ttest).

Results and Discussion:

While the average weight of prostate in animals from negative controlgroup was found to be 1.28 grams, and its organ coefficient was 0.34%,the average tissue weight and organ coefficient in animals from positivecontrol group, however, was found to be 0.73 grams and 0.23%,respectively. The average weight of prostate in animals from groupstreated with PEITC was found to be 0.79 g (oil base formulation at doseof 4 mg/kg), 0.91 g (oil base formulation at dose of 1 mg/kg), 0.83 g(aqueous base formulation at dose of 4 mg/kg) and 0.75 g (aqueous baseformulation at dose of 1 mg/kg), and their organ coefficient was 0.23%,0.25%, 0.23%, and 0.21%, respectively. They were significantly lowerthan the negative control group (P<0.05), demonstrating that the hormoneeffectively induced BPH and isothiocyanates, as exampled by PEITC ineither oil or aqueous preparation was able to inhibit the BPH, and theactivity was equal to the positive control drug, estradiol. This exampleindicated that PEITC was effective to inhibit BPH at the experimentaldose range.

Example 6 Therapeutic Effects of PEITC on BPH: Test Two

Materials and Methods

Reagents:

PETIC was the same as Example 2. Testosterone propionate, Proscar,ketamine, and penicillin were purchased from Wuxi Shanhe Health DrugChain Limited Participation Group.

Animals:

Adult pathogen-free SD rats, male, body weight ranging from 150 to 200grams, were purchased from the Animal Center of Nanjing MedicalUniversity. The animals were housed five per cage and fed ad libitum,fresh tap water and commercial rodent pellets. Animal rooms werecontrolled at 25±2° C. and a 12 hrs light/dark cycle.

Disease Model and Drug Treatment:

Thirty-six male SD rats were randomly divided into 4 groups, nine each.Group 1 was served as blank control group (pseudo-surgery control group)in which animals were under the same surgical procedures except notestis was sectioned and fed normally. Groups from 2 to 4 were served astreatment groups in which all animals were anesthetized using ketamineand castrated by removing both testises under sterile conditions. Afterthe surgery, the penicillin was injected to prevent the animals frominfection. One week later, the testosterone propionate was injectedsubcutaneously at dose of 5 mg/kg, once a day for a month. Group 2 wasserved as negative control in which animals were given saline orally fora month on the same day that the testosterone propionate was injected,once a day. Group 3 was served as positive control group in whichanimals were given Proscar by gavage at dose of 0.1 mg/rat/day for amonth beginning on the same day that the testosterone propionate wasinjected. Animals in the group 4 were orally treated with aqueous baseformulation of PEITC (Example 13-11) at dose of 2 mg/kg, once a day, fora month beginning on the same day that the testosterone propionate wasinjected.

Twenty-four hrs after the last dose, the animals were euthanized andweighed. Volume and weight of prostate were measured. The averages ofprostate volume/weight and organ coefficient (average weight ofprostate/average of body weight) were calculated, and statisticalanalysis performed (t test).

Histopathology:

Prostate tissues of animals from different groups were embedded withparaffin. The tissue sections were stained with H&E for histpathologicalexamination by an anatomical pathologist.

The experimental data were shown in Table 1&2.

TABLE 1 Inhibitory effects of PEITC on androgen-induced BPH in ratsAverage Inhibition of Average Body Average Weight Volume of Organ OrganCoefficient Weight (g) of Prostate (g) Prostate (cm³) Coefficient (%)(%) Blank Control 415 ± 14.1 0.78 ± 0.07 0.87 ± 0.12 0.187 ± 0.01 Negative 330 ± 22.6 1.41 ± 0.14 1.68 ± 0.17  0.43 ± 0.004 ControlPositive 340 ± 30.5 1.30 ± 0.12 1.61 ± 0.16 0.38 ± 0.05 11.63 Control(Proscar, 0.5 mg/kg) PEITC 337 ± 23.7 1.20 ± 0.16 1.42 ± 0.16 0.36 ±0.06 16.28 (2 mg/kg)

TABLE 2 Hispathological scores of BPH in animals from different groups.Connective tissue Enlargement proliferation, or Acinar of Lumen ofSecretions of papillary No. of Density Gland Gland hyperplasia AverageGroup Animals (Scores/No.) (Scores/No.) (Scores/No.) (Scores/No.) X ± SDScores^(a) 0 + ++ +++ 0 + ++ +++ 0 + ++ +++ 0 + ++ +++ Negative 9 1 2 60 1 3 5 0 2 2 5 0 6 2 1 0 4.78 ± 1.64 Control Positive 9 1 2 6 0 3 3 3 05 2 2 0 8 1 0 0 3.33 ± 1.11* Control (Proscar) PEITC 9 7 2 0 0 3 6 0 0 35 1 0 8 1 0 0 1.78 ± 1.09** ^(a)0, +, ++, and +++ represent score 0, 1,2, and 3 respectively. *P < 0.05 as compared with negative control; **P< 0.01 as compared with negative control

Results and Discussion:

Based on the comparison of the data of the blank control group and thoseof the negative control group, the rats of the negative control grouphad obvious prostate hyperplasia, indicating that BPH rat model wasvalid as shown in Table 1. Also shown in Table 1, treatment of Proscarand PEITC can reduce the volume of the prostate. Since BPH is a chronicdisease, we speculated that a better efficacy could be achievable if theBPH animals are treated with either Proscar or PEITC for longer periodof time, not just one month. The data of the weight and volume ofprostate, organ coefficient showed that both Proscar and PEITC wereeffective. From the comparison of the efficacy data between Proscar andPEITC, we found that PEITC might have better therapeutic activity thanProscar, which needs to be further confirmed by more experiments.

The histopathological examinations showed that major histopathologicalchanges in the BPH of the tested rats include abnormal acinar density,enlargement of lumen of gland, abnormal secretions of gland, togetherwith connective tissue proliferation and papillary hyperplasia. As shownin Table 2, FIG. 5, FIG. 6.1-6.5, the pathological changes of theanimals treated with PEITC and Proscar were significantly less severethan the negative control group (Proscar, P<0.05; PEITC P<0.01). Theywere similar to the data of the weights and volumes of the prostate, aswell as the organ coefficients indicated that the pathological changesof the animals treated with PEITC was not so obvious compared withProscar control group.

Example 7 Therapeutic Effects of PEITC on Non-Bacterial Prostatitis

Materials and Methods

Reagents:

PEITC was the same as Example 2. Xiaozhiling injection (Tannin potassiumaluminum sulfate injection) was purchased from Jinan YongningPharmaceutical Co., Ltd. GENURIN (Flavoxate Hydrochloride), ketamine,and penicillin were purchased from Wuxi Shanhe Health Drug Chain LimitedParticipation Group.

Animals:

Adult pathogen-free SD rats, male, body weight ranging from 150 to 200grams, were purchased from the Animal Center of Nanjing MedicalUniversity. The animals were housed five per cage and fed, ad libitum,fresh tap water and commercial rodent pellets. Animal rooms werecontrolled at 25±2° C. and a 12 hrs light/dark cycle.

Disease Model and Treatment:

Fifty-five male SD rats were randomly divided into 6 groups, 9 each,except the negative control group for which 10 animals were used. Allanimals from group 1 to 6 were anesthetized by injection of ketamineunder sterile conditions and incised around 1.5 cm with a sterilizedsurgery scalpel at middle of abdomen. Animals in Group 1 were injectedpenicillin to prevent infection, and given distill water, 10 ml/kg, oncea day, 5 times a week for 5 weeks. Groups from 2 to 6 injected 25%Xiaozhiling into left and right lobes of prostate, 0.1 ml for each lobeand sew the muscle and skin with #1 silk thread. The animals were thengiven penicillin to prevent infectious. Group 2 was served as negativecontrol group in which animals were treated with distill water by gavage24 hrs after the Xiaozhiling injection, once a day, 5 times a week for 5weeks. Group 3 was served as positive control group in which animalswere treated with GENURIN by gavage 24 hrs after the Xiaozhilinginjection at dose of 60 mg/kg, once a day, 5 times a week (escapingweekend) for 5 weeks. Groups 4 to 6 were treated by gavage with 4, 2,and 1 mg/kg of PEITC formulation described in Example 13-11 for the sameperiod of time as positive control group (once a day, 5 times a week for5 weeks). On the last dosing day, urine samples from different groupsthat covered periods of 1, 2 and 3 hrs were collected.

Twenty-four hrs after the last dose, the animals were euthanized andweighed. Volume and weight of prostate were measured. The averages ofthe prostate weight/volume and organ coefficient (average weight ofprostate/average of body weight) were calculated, and statisticalanalysis performed (t test).

Histopathology:

Prostate tissues of animals from different groups were embedded withparaffin. The tissue sections were stained with H&E for histpathologicalexamination by an anatomical pathologist.

Results and Discussion

The results were summarized in Table 3 below.

TABLE 3 Inhibitory effect of PEITC on enlarged prostate. Average of BodyInhibition of Weight Average Weight Average Volume of OrganOrgan-coefficient (g) of Prostate(g) Prostate (cm³) Coefficient(%) (%)Blank Control 417 ± 13.8 0.77 ± 0.05 0.85 ± 0.14 0.185 ± 0.02  NegativeControl 399 ± 14.4 1.03 ± 0.14 1.10 ± 0.20 0.260 ± 0.04  PositiveControl 356 ± 25.9 0.71 ± 0.09 0.69 ± 0.10 0.196 ± 0.03* 24.62 (GENURIN60 mg/kg) PEITC (1 mg/kg) 377 ± 39.1  0.7 ± 0.15 0.78 ± 0.16 0.186 ±0.03* 28.46 PEITC (2 mg/kg) 360 ± 24.8 0.70 ± 0.12 0.82 ± 0.13 0.188 ±0.03* 27.69 PEITC (4 mg/kg) 370 ± 24.8 0.72 ± 0.16 0.78 ± 0.10 0.192 ±0.03* 26.15 *P < 0.05 as compared with negative control.

TABLE 4 Diuretic effect of PEITC on animals with prostatitis 1 h Period(ml) 2 h Period (ml) 3 h Period (ml) Blank Control 3.5 ± 1.2  7.8 ± 1.78.1 ± 1.7 Negative Control 3.6 ± 1.6  8.0 ± 1.7 8.1 ± 1.9 PositiveControl 6.2 ± 1.3* 7.9 ± 1.9 8.8 ± 1.2 (GENURIN 60 mg/kg) PEITC (1mg/kg) 5.5 ± 1.0* 7.0 ± 0.7 7.9 ± 1.0 PEITC (2 mg/kg) 6.6 ± 2.1* 8.7 ±1.7 9.2 ± 2.7 PEITC (4 mg/kg) 5.7 ± 2.2* 9.4 ± 1.5 10.4 ± 1.9* *P < 0.05as compared with negative control

TABLE 5 Results of histophatological examination of SD rats prostatitisChronic No. of Inflammatory Inflammatory Group Animal Congestion EdemaInfiltration Infiltration Scores^(a) 0 + ++ +++ 0 + ++ +++ 0 + ++ +++0 + ++ +++ Negative 10  4 3 3 0 6 1 3 0 7 1 2 0 9 1 Control 0 0 GENURIN9 4 5 0 0 6 2 1 0 7 2 0 0 5 4 0 0 PEITC 9 7 2 0 0 5 4 0 0 7 2 0 0 9 0 (4mg/kg) 0 0 PEITC 9 4 5 0 0 6 3 0 0 6 3 0 0 8 0 (2 mg/kg) 1 0 PEITC 9 3 60 0 3 6 0 0 9 0 0 0 9 0 (1 mg/kg) 0 0 Overall Fibrous Average GroupBleeding Exudation Hyperblastosis X ± SD Scores^(a) 0 + ++ +++ 0 + +++++ 0 + ++ +++ Negative 5 4 1 0 3 3 7 3 0 0 4.20 ± 1.87 Control 3 1GENURIN 6 3 0 0 8 1 8 1 0 0 1.83 ± 0.75** 0 0 PEITC 6 3 0 0 4 4 9 0 0 01.72 ± 0.79** (4 mg/kg) 1 0 PEITC 8 1 0 0 5 4 7 2 0 0 2.16 ± 1.52** (2mg/kg) 0 0 PEITC 7 2 0 0 5 4 7 2 0 0 1.83 ± 1.06** (1 mg/kg) 0 0 ^(a)0,+, ++, and +++ represent score 0, 1, 2, and 3 respectively **P < 0.01 ascompared with negative control.

As shown in Table 3, PEITC effectively inhibited the prostateenlargement caused by prostatitis. This effect however, did not closelyrelate to dosages indicating that the lowest dose may already reach theoptimal activity. On the other hand, PEITC showed significant diureticeffect in a dose-dependent manner. The higher dose, the better diureticeffect was seen (Table 4). Diuretic effect of PEITC at dose of 4 mg/kgachieved statistically significance (P<0.05) as compared with untreatedcontrol, and this therapeutic effect was found to be better than that ofpositive control drug, GENURIN. Importantly, histopathologicalexaminations showed that all histopathological grades from animalstreated with either PEITC or the positive control drug, GENURIN werestatistically lower than that of untreated control (P<0.01). These datafurther provided solid evidence that PEITC was equal to GENURIN in termsof reduction of symptoms and histopathological lesions of prostatitis(Table 5 and FIG. 5, 7.1-7.5).

Example 8 Effects of Isothiocyanates on Human Prostate Cancer CellsGrowth In Vitro

Materials and Methods

Reagents:

Phenethyl isothiocyanate (PEITC) was the same as Example 2. Otherisothiocyanates, i.e. benzyl isothiocyanate (BITC), allyl isothiocyanate(AITC), 4-sulfophenylisothiocyanate (SPITC), were purchased fromSigma-Aldrich (USA). The working solutions of the above isothiocyanateswere prepared in DMSO except SPITC for which serum-free RPMI 1640 mediumwas used as a solvent.

Cell Culture:

The same procedures were used as described in Example 2.

Determination of Cell Growth:

MTT and SRB method as described in the reference (Kreis, Budman et al.1997). Briefly, human prostate cancer cells grown exponentially werealiquoted into 96-well plates at a density of 5000 cells/200 μl perwell. Twenty-four hrs after the incubation, the cells were exposed forthree or seven days to serial dilutions of indicated isothiocyanate.After the incubation, 100 μl of the medium was removed from each of thewells and 50 μl solution of3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wasadded, the cells were incubated for an additional 4 hrs and then 200 μlof 0.04 N HCl-isopropanol was added to each well to dissolve the blackformazan precipitates. The absorbance was measured at wavelength 540 nm.For SRB method, remove the medium after 72 hrs. The cells on 96-welldishes were fixed with 10% trichloride acetic acid for 1 hr, air-driedfor 24 hrs. The cells were then stained with 50 μl of SRB(sulforhodamine B) for 20-30 mM. After extensive washes with 1% aceticacid for 5 times, air-dried, the purple protein-SRB complex wasdissolved in 200 μl 10 mM Tris-HCl buffer (pH 10.0), and the absorbancewas measured at the wavelength of 540 and 630 nm. The percent of cellsurvival (T/C%) were calculated based on the results of test groups tothat of the control group. All of the test data were summarized in thefollowing Table 6. The semi-exponential curves of the percent of cellsurvival (T/C%) to the drug concentrations (FIG. 8) and IC₅₀ wereobtained by using Sigma-plot program.

TABLE 6 Inhibitory effects of isothiocyanates on the growth of humanprostate cancer cell line LNCaP Percent of Cell Survival (T/C %)Concentrations (μM) PEITC BITC SPITC AITC 0.0390 102.60 94.09 99.5498.84 0.0780 102.10 95.64 101.43 98.92 0.1562 88.10 97.91 101.43 98.260.3125 82.25 95.82 99.66 98.55 0.6250 82.25 71.25 98.26 97.10 1.250078.12 27.95 101.56 99.49 2.5000 30.24 14.83 104.37 83.95 5.0000 10.065.07 100.60 47.60 10.0000 1.52 0.88 101.33 41.01 20.0000 0.56 0.94 99.8838.69Results and Discussion

Using both MTT and SRB, we examined the effects of variousisothiocyanates (PEITC, BITC, AITC and SPITC) on human prostate cancercell LNCaP growth and compared their activities. We found that alltested isothiocyanates except SPITC were able to significantly inhibitedprostate cancer cell growth within the tested concentrations (0.039 to20 μM). PEITC and BITC showed similar growth inhibitory activity with anIC₅₀ between 0.8 to 1.5 μM, but AITC was relatively weaker with an IC₅₀approximately 10 μM (FIG. 8). These observations suggest that the growthinhibition on prostate cancer cells or on other malignant cells is oneof common biological or pharmacological characteristics ofisothiocyanates. We also observed as described in Example 11, that muchhigher concentration was needed for PEITC to achieve IC₅₀ on growthinhibition of B16 mouse melanoma cell line (IC₅₀ was approximatelybetween 10-20 μM). The inhibitory effect on the growth of human prostatecancer cells was stronger (8-10 times) than B16 cells. These resultssuggest that human cancer cells are more sensitive to isothiocyanatesthan animal cancer cells or human prostate cancer cells are moresensitive than other cancer cells.

Example 9 Growth Inhibitory Effects of PEITC on Hormone-Dependent andIndependent Prostate Cancer Cells

Materials and Methods

Reagents:

Phenethyl isothiocyanate (PEITC) was the same as Example 2. Otherchemicals were purchased from Sigma-Aldrich (USA), otherwise asindicated.

Cell Culture: Human Prostate Cancer Cell Line LNCaP.

The same procedures were used as described in Example 3.

MTT: MTT and SRB method were used to determine the effects of variousisothiocyanates on cell growth as described previously (Kreis, Budman etal, 1997). Briefly, human prostate cancer cell line LNCaP grownexponentially were aliquoted into 96-well plates at a density of 2500cells/200 μl per well. Twenty-four hrs after the incubation, the cellswere exposed for seven days to serial dilutions of indicatedisothiocyanate or paclitaxel. After the incubation, 100 μl of the mediumwas removed from each of the wells and 50 μl of a 1 mg/ml solution of3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wasadded, the cells were incubated for an additional 4 hrs and then 200 μlof 0.04 N HCl-isopropanol was added to each well to dissolve the blackformazan precipitates. The absorbance was measured at wavelength 540 nm.The percent of cell survival (T/C%) were calculated based on the resultsof test groups to that of the control group. The semi-exponential curvesof the percent of cell survival (T/C%) to the drug concentrations andIC₅₀ were obtained by using Sigma-plot program.

Results and Discussion

Since the overactivated androgen-androgen receptor (AR) is an importantfactor involved in the uncontrolled prostate and prostate cancer cellproliferation, and is as important as, might much important to hormoneindependent prostate cancer cell. thus, blockage of the translation andexpression of AR is expected to suppresses growth of bothhormone-dependent and independent prostate cancer cell. Therefore, inthis experiment, we used MIT method to examine the inhibitory effects ofPEITC on prostate cancer cell growth, and compared the activity betweenhormone dependent (LNCaP AD) and hormone independent (LNCaP AI) celllines. As shown in FIG. 9B, a significant protate cancer cell growthinhibition of PEITC was observed. More importantly, an almost equal IC₅₀(0.6 μM) was observed in both AD and AI cell lines. Under the sameexperimental conditions, paciltaxel, a clinical widely used anticancerdrug also achieved significant growth inhibition. However, when wecompared the IC₅₀ between AD and AI in response to paclitaxel, we foundthat, in contrast to PEITC, AI cells showed significant resistance topaclitaxel as indicated by 60 times higher of IC₅₀ (IC₅₀ was found to be0.01 nM for AD and 0.6 nM for AI cells, FIG. 9A). The results were inaccordance with those we observed before. The data also demonstratedthat the activity of paclitaxel was much higher than that of PEITC, butgiven the fact that paclitaxel has high toxicities while it achievestherapeutic effects. For example, when paclitaxel plasma concentrationis at 0.05 μM, it will produce severe bone marrow suppression. Incontrast, PEITC is present in natural food, and has lowertoxicities/side effects. The clinical data has demonstrated thatpaclitaxel could not prolong the life of the patients of the post stageof prostate cancer besides its toxicities.

Example 10 Anti Cancer Activities In Vivo and Effects on AR Expressionof PEITC-NAC

Materials and Methods

Reagents:

PEITC-NAC was the same as Example 2. Other chemicals were purchased fromSigma-Aldrich (USA), otherwise as indicated.

Xenograft Tumor and Treatment:

Immunodeficient BALB/c nude mice, male, 5 weeks old, were purchased fromAnimal Center, Chinese Academy of Sciences, Shanghai, China. The animalswere housed in an SPF animal lab. Human androgen independent prostatecancer PC-3 cells, in the form of a pellet (approximately 1×106 cellsper animal), were mixed with a 50% volume of Matrigel and implanted s.c.in the flank of the animal. Twenty-four hrs after the transplant, themice were randomly divided into two groups, eight animal each. One groupserved as untreated control and fed with regular AIN76 diet, and anotherone as treatment group in which AIN76 diet containing 8 μM/g ofPEITC-NAC was provided. The animals were treated for 30 days, and tumorincidence and size were measured at day 7, 10, 22. Twenty-four hrs afterthe last administration, animals were euthanized, and body and tumorswere weighted. The percentages of the turner inhibition were calculatedand, the results were shown in Table 7. Western blot was used to followthe possible change of AR expression.

TABLE 7 Anticancer activities of PEITC-NAC on xenograft hormoneindependent human prostate cancer cell line PC-3 Untreated ControlPEITC-NAC at Different Days Days After No. of No. of No. of No. of TumorAnimals Animals Incidence Animals with Animals Incidence InhibitionInplantation with Tumor without Tumor (%) Tumor without Tumor (%) (%) 77 1 87.5 6 2 75 — 10 8 0 100 6 2 75 — 22 8 0 100 7 1 87.5 — At Terminal8 0 100 7 1 87.5 53.3Results and Discussion

Immunodeficient xenograft tumor models were widely used to evaluatetherapeutic activities of new potential anticancer drugs. In order toexamine the possible anticancer activity of PEITC-NAC, xenograft modelof human androgen independent PC-3 prostate cancer cells was used. Asshown in Table 7, PEITC-ANC effectively inhibited PC-3 tumor growth by53.3% (P<0.02) at given dose. More interestingly, one animal wascancer-free.

Example 11 Growth Inhibitory Effects of PEITC on Mouse B16 Melanoma InVitro

Materials and Methods

Reagents:

PEITC was the same as Example 2. Mouse B16 melanoma cell line waspurchased from the Shanghai Institute of Cell Biology, Chinese Academyof Sciences, Shanghai, China. Fetal bovine serum (FBS) was purchasedfrom Shanghai Hua-Mei Biochemical Reagents Company. RPMI 1640 medium waspurchased from Gibco. Penicillin and streptomycin were medical products.Glutamine and trypan blue were provided by Sigma Company. Trypsin wasfrom Amresco. 24-well cell culture plates were provided by CostarCompany (US). Dimethyl sulfoxide (DMSO) was purchased from ShanghaiMedical/Shanghai Chemical Reagents Company.

Cell Culture:

RPMI 1640 medium was adjusted to pH7.0˜7.2 by using 1 mol/L HCl or NaOH,sterilizing filtration, then kept at 4° C. in a refrigerator. Beforeusing, 10% FBS, 1% 200 mmol/L glutamine, 100 U/ml Penicillin and 100U/ml streptomycin were added in to make a working medium. B16 mousemelanoma cells were cultured in the medium at 37° C. with saturatedmoistures in 5% CO₂ incubator.

Determination of Cell Growth Curve:

B16 mouse melanoma cells at exponential growth phase were digested by0.25% trypsin, adjusted to the density of 2˜5×10⁴/mL by adding the aboveworking RPMI 1640 medium, sed into 24-well dishes at 1 ml per well,under normal culture conditions mentioned above. Twenty-four hrs afterthe incubation, the cells were exposed to 5, 10, and 15 μM of PEITC intriplet, respectively. Equal volume of the working RPMI 1640 medium wasadded into the wells of the control group. The cell numbers from eachwell were counted in triple using trypan blue exclusion method from day0 to day 5. The cell growth curve was obtained by plotting the mean ofcell numbers at each time points with the time (FIG. 10).

Determination of Cell Growth Inhibition:

B16 mouse melanoma cells at exponential growth phase were digested by0.25% trypsin, adjusted to the density of 3˜5×10⁴/mL by adding the aboveworking RPMI 1640 medium, sed into 96-well dishes at 150 μl per well,under normal culture conditions mentioned above. Twenty-four hrs afterthe incubation, the cells were exposed for 48 h at 37° C. with saturatedmoistures in 5% CO₂ incubator to different concentrations of PEITC.Equal volume of the working RPMI 1640 medium was added into the wells ofthe control group. Each group had four wells. After incubation,discarded the supernatant, added 150 μl D-Hanks per well, washed,discarded the supernatant. After adding 100 μl MTT (0.5 mg/mL),incubated for another 4 hrs. Discarded the supernatant, added 150 μlDMSO, kept at 37° C. for 10 mins. Absorbance was measured at wavelengthof 570 nm. The cell growth inhibition was shown in Table 8.

TABLE 8 Inhibitory effects of PEITC on B16 melanoma cells Concentrationsof PEITC (μmol/L) Growth Inhibition (%) Blank Control / 2.5 14.46 5.027.93 7.5 36.67 10.0 49.23 12.5 58.41 15.0 63.69 20.0 77.35 30.0 89.6440.0 96.83Results and Discussion:

PEITC showed a significant growth inhibition of B16 mouse melanoma cellsin a concentration dependent manner as demonstrated by the data in Table8 and FIG. 10. The higher concentration, the stronger growth inhibitionwas seen from the inhibition results. It reached over 96% growthinhibition at concentration of 40 μM. These experiments thusdemonstrated that PEITC was effective against B16 melanomaproliferation.

Example 12 Inhibitory Effects of PEITC in Xenograft B16 Mouse Melanoma

Materials and Methods

Reagents:

PEITC was the same as Example 2. B 16 mouse melanoma cell line waspurchased from the Shanghai Institute of Cell Biology, Chinese Academyof Sciences, Shanghai, China. Fetal bovine serum (FBS) was purchasedfrom Shanghai Hua-Mei Biochemical Reagents Company. RPMI 1640 medium waspurchased from Gibco. Penicillin and streptomycin were medical products.Sigma Company provided glutamine and Trypan blue. Trypsin was fromAmresco. Cyclophosphamide (CTX) for injection was purchased from JiangsuHengrui Medicine Co., Ltd. Saline was purchased from Shanghai ChangzhengFumin medicine Tongling Co., Ltd.

Animal:

Healthy male Kunming mice, at age of 4 to 6 weeks, body weight 18 to 22grams, were purchased from the Animal Center of Nanjing MedicalUniversity.

Cell Culture, Xenograft B16 Mouse Tumor, and its Passage In Vivo:

B16 mouse melanoma cells were maintained in RPMI 1640 medium containing10% FBS, 1% 200 mmol/L glutamine, supplemented with 100 U/ml Penicillinand 100 U/ml streptomycin at 37° C. with saturated moistures in 5% CO₂incubator. Adherent cells were digested by 0.25% trypsin for passaging.The B16 mouse melanoma cells at exponential growth phase were suspendedin PBS. The cell viability was examined using trepan blue exclusionmethod to ensure the viability above 98%. Adjusted the suspension to acell concentration of approximately 1×10⁷/ml. 0.2 ml of the suspensionwere transplanted subcutaneously into right axillary subcutis forpassaging.

Treatment of Xenograft B16 Tumor with PEITC:

Under sterile condition the xenograft B16 mouse tumor in mice wasstripped from axillary subcutis. After cutting by scissors, the tumorwith saline (1:3) was ground in a glass homogenizer. Monocell suspensionwas made with saline. The cell viability was examined using trepan blueexclusion method to ensure the viability above 98%. Then adjusted thesuspension to a cell density of approximately 1×10⁷/ml with saline. 0.2ml were transplanted subcutaneously into right axillary subcutis of eachmouse. After transplanting, randomly divided the mouse into 5 groups, 12each.

Started from the next day the animals were treated with or withouttested article or control articles once a day for 15 days as follows:two groups were given two different doses of PEITC composition (70 and120 mg/kg of PEITC, the API, composition as described in Example 13-16)orally by gavage; one group was served as negative control in whichanimals were given saline only by gavage; another group was served aspositive control in which animals were given 20 mg/kg ofcyclophosphamide via i.p. Twenty-four hours after the lastadministration, the animals were euthanized. Tumors were taken out,sizes and weights were measured, and the percent of inhibitions werecalculated. The data were shown in Table 9.

Measurement of Tumor Growth Curve:

Tumor volume was measured according to the method described in theofficial book of Ministry of Health, Drug Council “Principals ofPreclinicl Research of New Drug (Western Medicine)”. The measurementbegan at day 4 after the tumor transplantation. The long trail (A) andshort track (B) were measured with a vernier caliper, and repeated 3times for each measurement. The tumor volume was then calculated by thefollowing equation:V=π/6[(A+B)/2]³Where “A” represents a long trail and “B” represents a short track.A tumor growth curve was obtained by plotting the tumor volume with time(FIG. 11).

TABLE 9 Inhibitory effects of PEITC on xenograft B16 mouse tumor Changesin Average No. of Dose Body Weight of Tumor Inhibition Route GroupAnimal (mg/kg/d) (g) Weight (g) (%) Negative Control 12 / 10.32 ± 2.37 2.31 ± 0.85 / O.P. PEITC (70 mg/kg) 12 70 9.78 ± 4.28 1.23 ± 0.68* 39.23PEITC (120 mg/kg) 12 120   6.34 ± 3.42* 0.91 ± 0.23**∇ 58.54 i.p. CTX(20 mg/kg) 12 20  5.07 ± 2.64* 0.76 ± 0.41** 66.23 *P < 0.05 as comparedwith the negative control **P < 0.01 as compared with the negativecontrol ∇P > 0.05 as compared with the positive controlResults and Discussion:

(1) Compared with the negative control, treatment of xenograft B16 mousetumor with PEITC by gavage achieved significant anticancer activities(P<0.05) as shown in Table 9. Also the anticancer efficacy of PEITC wasdose-dependent under the test condition. There is no significantdifference (P>0.05) between the 58.54% inhibition on xenograft B16 mousetumor with PEITC and 66.23% inhibition on xenograft B16 mouse tumor withcyclophosphamide (CTX). PEITC is effective via oral administration, thusit is more convenient to patients than the CTX via injection.

(2) FIG. 11 shows that tumor volume is parallel very well with the tumorweight reduction when the tumor volume growth curves is used as theindication for the tumor inhibition. The results confirmed that thePEITC via gavage inhibited the xenograft B16 mouse melanoma, and theefficacy was dose-dependent.

From table 9 and FIG. 11, conclusion can be summarized: PEITC showsanticancer activities on the xenograft B16 mouse melanoma Since melanomais the most harmful skin cancer in human, our observations in thisinvention demonstrated that PEITC is effective against the xenograft B16mouse melanoma, it is reasonable to assume that PEITC will be effectiveagainst human skin cancer.

Example 13 Studies of Different Formulations of PEITC

In this invention, we widely, deeply and rationally screened varietiesof surfactants, lipid ingredients, and other excipients in order toformulate a better stable and bioavailable isothiocyanate compositionfor pharmaceutical products and dietary supplements. The in vivo studydemonstrated that these compositions have effective inhibition on benignprostatic hyperplasia, prostatitis and skin cancer.

In this invention, the ingredients of the composition for pharmaceuticalproducts and dietary supplements include:

-   -   (a) natural or synthetic isothiocyanates as API;    -   (b) surfactants or solubilizing agent: The surfactant used in        this invention was to emulsify or enhance solubility of        isothiocyanates. Since a surfactant contains both hydrophilic        and lipophilic groups, it can surround the API molecules, thus        to emulsify and enhance solubility of API and increase their        stability;    -   (c) lipid ingredients to be used as diluents or a cosolvent;    -   (d) antioxidants: to prevent oxidation damage;    -   (e) other excipients: such as absorbent, diluent, lubricant,        binder, disintegrant, solvent, covering and coating material        etc. Application of these components in this patent would allow        us to make different kinds of formulations like tablets,        granules, pills, lyophilized powers, solutions, emulsion,        injections, pastes, films, suppositories, sprays, implants and        all other available dosage forms.

All formulations in this invention containing PEITC are used aspharmaceutical products or dietary supplements, which are effectiveagainst benign prostatic hyperplasia, prostatitis and skin cancer.

Example 13-1

4-sulfophenylisothiocyanate (SPITC) 10 mg Microcrystalline cellulose(PH102) 90 mg Anhydrous lactose 150 mg  Colloidal silicon dioxide  2 mg

Procedure:

Weighed formulation amount of 4-sulfophenylisothiocyanate, addedproportional amount of microcrystalline cellulose (PH102) and anhydrouslactose on the basis of contour incremental principle. Then addedcolloidal silicon dioxide, mixed evenly, and directly compressed to maketablets.

4-sulfophenylisothiocyanate is a power with an excellent dispersiveproperty. Due to the relative low amount of 4-sulfophenylisothiocyanatein this formulation, microcrystalline cellulose and anhydrous lactosehave been used as diluents and directly compressed to tablets.

Example 13-2

4-sulfophenylisothiocyanate(SPITC)  10 mg Starlac 288 mg Magnesiumstearate  2 mg

Procedure:

Mix starlac with 4-sulfophenylisothiocyanate for 5 min. After addingmagnesium stearate, mix for additional 5 min, Compress the mixturedirectly to make the tablets.

Since the starch is inside and lactose outside, the starlac hasexcellent anti-moisture property. Based on this character, we choosesatrlac as diluents in this formulation and compress directly to maketablets.

Example 13-3

Phenethyl isothiocyanate (PEITC)  2.5 mg Medium chain triglyceride 350mg

Procedure:

Phenethyl isothiocyanate (PEITC) was added to medium chain triglyceridesand mixed evenly. The mixture was then used to prepare for soft capsulesor hard capsules.

Due to an almost water insoluble liquid, formulations are limited forphenethyl isothiocyanate. In the current formulation, the medium chaintriglyceride with relative short carbon chain is used because of itsgood dispersive nature to keep uniform and stability.

Example 13-4

Benzyl isothiocyanate (BITC)  2.5 mg Soybean oil 350 mg

Procedure:

Benzyl isothiocyanate (BITC) was added to soybean oil and stirred to mixevenly. The mixture was then used to fill soft capsules or hardcapsules.

As compared with Example 13-3, in this formulation, soybean oil was usedinstead of medium chain triglyceride since soybean oil is easy to obtainwith lower price. However soybean oil contains long chain carbon, it mayaffect the absorption of the API.

Example 13-5

Phenethyl isothiocyanate (PEITC)  2.5 mg Vitamin E polyethylene glycolsuccinate 125 mg

Procedure:

Phenethyl isothiocyanate and vitamin E polyethylene glycol succinatewere heated at 60° C. to melt down, and then mixed evenly. The mixturewas used to fill hard capsules.

Vitamin E polyethylene glycol succinate is en excellent surfactant; itsignificant increases dissolution rate of the API when it is used as anexcipient. However, Vitamin E polyethylene glycol succinate containsbiological active alpha-tocopherol, thus, the potential side effects inthis formulation needs to be evaluated.

Example 13-6

Phenethyl isothiocyanate (PEITC) 2.5 mg Polyoxyethylene (40)monostearate  25 mg

Procedure:

Phenethyl isothiocyanate and polyoxyethylene (40) monostearate wereheated at 60° C. to melt down, and then mixed evenly. The mixture wasthen used to fill hard capsules of PEITC.

Polyoxyethylene (40) monostearate is one of classic surfactants used asa pharmacopeia listed excipient, and has no potential side effects ofbiological activity as vitamin E polyethylene glycol succinate. Asconfirmed in this invention, this formulation exhibited betterdissolution rate and stability.

Example 13-7

Benzyl isothiocyanate (BITC)  50 mg Polyethylene glycol 6000  300 mgPolyoxyethylene (40) monostearate 1200 mg

Procedure:

Polyoxyethylene (40) monostearate w as heated on heater with magneticstirrer to melt down; benzyl isothiocyanate (BITC) was then added andmixed evenly. The mixture was kept warm at 60° C. until no air bubbles,and dropped into coolant dimethyl silicone. The BITC dropping pills werethen obtained.

Example 13-8

Dropping Pill Recipe

Phenethyl isothiocyanate (PEITC)  50 mg Polyethylene glycol 6000  450 mgPolyoxyethylene (40) monostearate 1050 mg

Coating Recipe

Eudragit E100 100 mg  Acetone 1420 mg  Talc/magnesium stearate 50 mgPolyethylene glycol 6000(pass 80 mesh) 10 mg H₂O 20 mg

Procedure:

Suitable amount of polyethylene glycol 6000 (PEG6000) andpolyoxyethylene (40) monostearate indicated in above dropping pillrecipe were heated to melt down, PEITC was then added and mixed evenly.The mixture was kept at a 70° C. water bath until no air bubble. Andthen dropped the mixture into the coolant dimethyl silicone to formPEITC dropping pills.

Suitable amount of talc/magnesium stearate indicated in the coatingrecipe above was added to acetone and mixed evenly (acetone mixture).Suitable amount of PEG 6000 was dissolved in suitable amount H₂Oindicated in the coating recipe above (PEG solution). The acetonemixture was then mixed evenly with PEG 6000 solution and dispensed witha ball mill to make an uniform suspension (PEG suspension). Suitableamount of Eudragit E100 was dissolved in acetone to obtain 12.5%Eudragit acetone solution. Add this solution to the PEG suspension.Adjust the newly formed mixture with acetone to obtain a 8-10% coatingsolution.

The PEITC dropping pills were placed into coated pot, and the coatingsolution was sprayed while the pot was rotated, and the pills coated.PEITC capsule was then obtained by filling empty capsules with suitableamount of coated PEITC dropping pills.

Example 13-9

Phenethyl isothiocyanate (PEITC) 25 mg Vitamin E polyethylene glycolsuccinate 50 mg Poloxamer F-127 50 mg Sorbitol 75 mg Porous starch 193mg Microcrystalline cellulose PH101 392 mg 10% polyvinylpyrrolidone K30alcohol solution 0.5 ml Talc 15 mg

Procedure:

Suitable amount of PEITC, vitamin E polyethylene glycol succinate andpoloxamer F-127 specified in recipe above were heated to melt down. Thesorbitol, porous starch and microcrystalline cellulose PH101 was added,stirred until mixing evenly. 10% polyvinylpyrrolidone K30 alcoholsolution was added while stirring to make granules, passed sieve, anddried at a 60° C. oven for 30 min to get dry granules. The talc was thenadded, stirred until uniform and hard capsules were filled.

Example 13-10

Benzyl isothiocyanate (BITC) 2.5 mg Medium-chain triglycerides   2 mgTween 80  25 mg

Procedure:

Suitable amount of BITC, medium-chain triglycerides and Tween 80specified in above recipe were mixed evenly, and the mixture was used tofill hard capsules. In order to improve the dissolution rate of BITC,add the surfactant Tween 80 to the recipe in example 13-3. Experimentaldata showed that the dissolution rate was significantly improved.

Example 13-11

Phenethyl isothiocyanate (PEITC) 2.5 mg Medium-chain triglycerides   2mg Polyoxyethylene (40) monostearate  25 mg

Procedure:

Suitable amount of polyoxyethylene (40) monostearate specified in aboverecipe were melted at 60° C., mixed well with PEITC and medium-chaintriglycerides. The mixture was used to fill hard gel capsules.

The products made according to this formulation were used forexperiments described in Example 5 (Therapeutic Effects of PEITC on BPH:Test One) Example 6 (Therapeutic Effects of PEITC on BPH: Test Two) andExample 7 (Therapeutic Effects of PEITC on Non-bacterial Prostatitis).Satisfactory results were obtained from all of these experiments.

The design of formulations of PEITC were restricted due to its poorsolubility in aqueous solution. Referring example 13-3 and example 13-6,both medium-chain triglycerides and polyoxyethylene (40) monostearatewere used at the same time in this formulation. Polyoxyethylene (40)monostearate has been widely used as the surfactant in the formulationsof poor solubility API. Our experiment support the conclusion thatpolyoxyethylene (40) monostearate enhances the solubility of APIotherwise it would be difficulty to dissolve. Experiments both in vitroand in vivo showed that this formulation was not only rather stable, butalso had a better dissolution rate. Thus, we conclude that thisformulation may be proven to be one of the valuable formulations ofPEITC in clinical application.

Example 13-12

Allyl isothiocyanate (AITC)  2.5 mg Medium-chain triglycerides 320 mgPolyoxyethylene (40) monostearate  40 mg

Procedure:

Suitable amount of allyl isothiocyanate (AITC), medium-chaintriglycerides, and Polyoxyethylene (40) monostearate specified in aboverecipe were heated to melt, stirred and mixed evenly. The mixture wasthen pressed to make soft capsules.

Since allyl isothiocyanate also is a liquid with poor solubility inwater, the same principle described in Example 13-11 was applied. Toincrease the stability of AITC, a slight higher percentage ofmedium-chain triglycerides was used in this formulation.

Example 13-13

Phenethyl isothiocyanate (PEITC)  50 mg Stearic acid  150 mgPolyoxyethylene (40) monostearate 1350 mg

Procedure:

Stearic acid and polyoxyethylene (40) monostearate was melted by heatingon a heater with magnetic stirrer, the PEITC was then added whilestirring. The mixture was kept at 60° C. in a water bath until no airbubble, dropped into the coolant dimethyl silicone to obtain droppingpills of PEITC.

This formulation was also an improvement of the formulation of Example13-11. Compared with recipe in Example 13-11, stearic acid was used toreplace medium-chain triglycerides. The idea is that stearic acid isstructurally similar to polyoxyethylene (40) monostearate, thus a bettercompatibility can be expected when these two are used together.

Example 13-14

Benzyl isothiocyanate (BITC)  50 mg Stearic acid  300 mg Polyoxyethylene(40) monostearate 1200 mg

Procedure:

Stearic acid and polyoxyethylene (40) monostearate was melted by heatingon a heater with magnetic stirrer, the BITC was then added whilestirring. The mixture was kept at 60° C. in a water bath until no airbubble, dropped into the coolant dimethyl silicone was to obtaindropping pills of BITC.

Compared with example 13-13, in this formulation the ratio of stearicacid to the API and polyoxyethylene (40) monostearate was increased,which increased melting point of the mixture and made it easier to formdropping pills besides the change of API from PEITC to BITC.

Example 13-15

Benzyl isothiocyanate (BITC)  25 mg Medium-chain triglycerides  20 mgPolyoxyethylene (40) monostearate 100 mg Vitamin E polyethylene glycolsuccinate 125 mg

Procedure:

BITC, medium-chain triglycerides, polyoxyethylene (40) monostearate, andvitamin E polyethylene glycol succinate were melted by heating to 60° C.on a heater. After mixing evenly, fill the mixture into hard capsules.

Example 13-16

Phenethyl isothiocyanate (PEITC) 25 mg Medium-chain triglycerides 20 mgPolyoxyethylene (40) monostearate 75 mg Polyoxyethylene (35) castor oil50 mg

Procedure:

PEITC, medium-chain triglycerides, polyoxyethylene (40) monostearate,and polyoxyethylene (35) castor oil were melted by heating to 60° C. ona heater. After mixing evenly, fill the mixture into capsules.

This formulation was modified from the formulation of Example 13-11, inwhich polyoxyethylene (35) castor oil was added to improve thesolubility of API in aqueous phase.

Example 13-17

Benzyl isothiocyanate (BITC) 25 mg Cottonseed oil 20 mg Polyoxyethylene(40) monostearate 250 mg  Polyoxyethylene (35) castor oil 50 mg

Procedure:

BITC, cottonseed oil, polyoxyethylene (40) monostearate, andpolyoxyethylene (35) castor oil were melted by heating to 60° C. on aheater. After mixing evenly, fill the mixture into hard gel capsules.

Example 13-18

Phenethyl isothiocyanate (PEITC) 100 mg Medium-chain triglycerides  90mg Polyoxyethylene (40) monostearate 250 mg Polyethylene glycol 60002500 mg 

Procedure:

Polyethylene glycol 6000 and polyoxyethylene (40) monostearate wereheated to melt on a heater with magnetic stirrer. PEITC and medium-chaintriglycerides were then added and mixed. The mixture was kept at 70° C.in a water bath until no air bubble, dropped into the coolant dimethylsilicone to obtain dropping pills of PEITC. The remaining dimethylsilicone on the surface of dropping pills was washed away with anhydrousether. Fill the mixture into capsules after the ether was evaporated.

Compared with the formulation of Example 13-11, the melting point ofthis formulation was increased as a result of the addition of highermelting point polyethylene glycol 6000, which made dropping pills easyto form.

Example 13-19

Phenethyl isothiocyanate (PEITC) 25 mg Vitamin E (tocopherol) 25 mgVitamin E polyethylene glycol succinate 75 mg Polyethylene glycol 400 25mg

Procedure:

Polyethylene glycol 4000 and vitamin E polyethylene glycol succinatewere warmed up to 60° C. on a water bath, PEITC and tocopherol were thenadded, mixed evenly, and filled into hard gel capsules when the mixturewas warm.

In this formulation, vitamin E polyethylene glycol succinate was used assurfactant. Since vitamin E polyethylene glycol succinate consists ofα-tocopherol and polyethylene glycol, on the basis of the principle ofsimilar structure, similar dissolvability the affinity among thesecomponents in this formulation would be better. However, the shortage ofthis formulation is that both vitamin E polyethylene glycol succinateand vitamin E themselves have biological activities, which may result inpositive or negative effects to specific diseases.

Example 13-20

Phenethyl isothiocyanate (PEITC) 2.5 mg  Medium-chain triglycerides 2.0mg  Polyoxyethylene (40) monostearate 25 mg Sodium carboxymethyl starch(CMS) 300 mg  Polyethylene glycol 6000 (pass 80 mesh) 10 mg

Procedure:

PEITC, polyoxyethylene (40) monostearate and medium-chain triglycerideswere warmed up to melt, sodium carboxymethyl starch (CMS) was thenadded, and mixed, followed by the addition of polyethylene glycol 6000powder. After mixing evenly, fill the mixture into capsules.

Sodium carboxymethyl starch (CMS) is an excellent binder. In thisformulation we intended to explore whether we could develop a powderformulation containing liquid form of PEITC based on the formulation ofexample 13-11.

Example 13-21

Tablet Recipe:

4-sulfophenylisothiocyanate (SPITC)  10 mg β-cyclodextrin (Kleptose DC)240 mg Mannitol (Pearlitol SD200) 240 mg Magnesium Stearte  10 mgCoating Recipe:

Hydroxypropylmethyl cellulose E15 0.4 mg Hydroxypropyl cellulose EF 0.4mg Polyethylene glycol 400 0.08 mg  Potassium sorbate 0.014 mg  H₂O  10mg

Procedure:

4-sulfophenylisothiocyanate (SPITC), Kleptose DC and Pearlitol SD200were added into the turbine agitator and agitated for 10 mins. MagnesiumStearte was then added, and agitated for additional 5 mins. The mixingpowder was directly compressed into tablets.

Adequate amount of water was warmed up, and added withhydroxypropylmethyl cellulose E15, hydroxypropyl cellulose EF,polyethylene glycol 400 and potassium sorbate while stirring to obtaincoating solution. The tablets obtained above were then placed into acoating pan. The coated tablets were obtained by spraying the coatingsolution on the tablets while rotating.

Example 13-22

Phenethyl isothiocyanate (PEITC)  25 mg Polyoxyethylene (40)hydrogenated castor oil 225 mg

Procedure:

PEITC was mixed with polyoxyethylene (40) hydrogenated castor oilevenly, and then filled into hard gel capsules at 40° C.

Polyoxyethylene (40) hydrogenated castor oil is one of popularsolubilizing agents for API with poor solubility recently. In additionto its efficiency, its semi-solid nature under room temperature makes itas an ideal excipent for semi-solid pharmaceutical preparations.

Example 13-23

Phenethyl isothiocyanate (PEITC) 25 mg Polyoxyethylene (35) castor oil200 mg  Tween 80 75 mg Vitamin E (tocopherol)  5 mg

Procedure:

PEITC was evenly mixed with polyoxyethylene (35) castor oil, Tween 80,and vitamin E (tocopherol), and the mixture was then filled into softgel capsules.

Example 14 Stability Testing of Isothiocyanate Compositions

Instruments:

S.C.101 electric thermostat dry oven, Zhejing Jiaxing XingshengElectrical Heating Instrument Factory; METTLER AE 100 electronicbalance, Ruishimeitele, Swiss; Agilent 1100 HPLC, Agilent, USA.

Method:

Suitable amount of API (isothiocyanates, as control) and compositionswere placed into clean vials, and sealed with rubber stoppers plusaluminum caps. All samples were stored at 60° C., and then assayed by avalidated HPLC method at time day 0, day 5, and day 10.

Results:

The test results were summarized in Table 10.

TABLE 10 Partial results of stability testing of isothiocyanateformulations Under 60° C. Compositions in Example for 10 days (API %)Control (exampled by PEITC) 95.2 13-3 98.8 13-4 96.9 13-6 95.3 13-1199.4 13-12 99.0 13-21 95.4

Discussion:

The results shown above indicated that compositions composing ofisothiocyanates and oil components (example 13-3, 13-4) above was morestable than the API themselves. In addition, stability ofisothiocyanates in the mixtures of oil (corn oil or medium-chaintriglycerides) and polyoxyethylene (40) monostearate at different ratiosalso improved as compared with themselves alone. These observationssuggest that adequate oil excipients and surfactant polyoxyethylene (40)monostearate may increase the stability of isothiocyanates although themechanisms need to be defined.

Example 15 Testing of in Vitro Dissolution of IsothiocyanateCompositions

Materials and Methods:

Medicine: Isothiocyanates compositions were the same as described inexample 13.

Instruments: Dissolution tester: Smart Drug Dissolution Instrument,Tianjin University Precision Instrument Factory; HPLC: Agilent 1100HPLC, Agilent, USA.

Dissolution Measurement: Dissolution rates of the compositions weremeasured according to the procedures described in the ChinesePharmacopeia 2005, Appendix XC, Method 2: Paddle Determination. Briefly,samples are added into 500 ml of de-aired, and deionized water which iskept at 37±0.5° C. for 45 min while the paddle rotates at 200 r/min.Take samples and let the sample be passed through the 0.8 μm filtermembrane, and analyzed by HPLC. The dissolution rate was thencalculated.

Results:

The results were shown in Table 11.

TABLE 11 Partial results of dissolution rate of isothiocyanateformulations Compositions in Example Dissolution Rate % Control(exampled by PEITC) 0 13-7 73.9 13-8 62.9 13-11 65.0 13-13 57.3 13-1416.8 13-15 72.9 13-16 70.1 13-19 52.0 13-22 86.6 13-23 77.2

Experiment Discussion: The data shown in Table 11 indicated that thedissolution rate of isothiocyanate itself was nearly zero. However, whenit was formulated with various oil excipients plus surfactant(s), thedissolution rate of isothiocyanate was improved, and the oil componentssomehow were negatively related to the dissolution from the experimentresults.

On the basis of stability testing results shown in example 14 anddissolution testing data in example 15, we conclude that the stabilitiesof isothiocynates formulated with oil or formulated with both oil andsurfactant(s) are acceptable. On the other hand the dissolution ratesare better in compositions containing oil plus surfactant(s) than incompositions containing oil only. Moreover, the pharmacodynamic studiesdescribed in example 5, 6, 7, 10, and 12 using compositions containingexcipients of both oil and surfactants also showed significantlytherapeutic activities in preventing and treating benign prostateshyperlasia, prostatitis, prostate cancer and skin cancer.

Discussion:

Prostatic disease is one of major diseases that significantly impact thequality of men's life, particularly elder men, and the incidence of thedisease is increasing worldwide. Drug treatment is still the mainmethodology for the treatment of the disease at present. However,current drugs for prostatic diseases have unwanted or unfavorableeffects. For example, medications derived from steroid hormones havesome side effects of steroids. Other drugs are either lack of knowledgeof API, unclear mechanism, or their therapeutic efficacies are notsatisfactory. Thus, to develop new pharmaceutical products, dietarysupplements or cosmetic products with ideal efficacy and safety profilesfor the treatment and prevention of various prostatic diseases iswarranted.

The major risk factor of skin cancer is UV light which mainly comes fromthe exposure of sun, thus everyone, no matter his/her race, skinproperty, age, occupation, and residential location, has risk to developskin cancer. In recent years the incidence of skin cancer is constantlyincreasing as a result of more sun light exposure due to the popularityof outdoor activities which is one of the results of uprising livingstandard. While effective drug is limited, it will have important valueon the medical application to develop a pharmaceutical product ordietary supplement for the treatment and prevention of skin cancer.

In this invention, we demonstrated, via test data obtained from variousin vitro and in vivo models, that isothiocyanates which had theisothiocyanic function group, included but not limited to phenethylisothiocyanate (PEITC), benzyl isothiocyanate (BITC), allylisothiocyanate (AITC), and 4-sulfophenylisothiocyanate (SPITC), andN-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC) etc.,effectively treated and/or prevented prostatic diseases including butnot limited to benign prostatic hyperplasia, prostatitis, and prostatecancer. On the basis of above findings, we further studied molecularmechanisms by which isothiocyanates effectively treat and preventprostatic diseases. We showed that isothiocyanates induce expression ofphase II detoxification enzyme GSTP1. The increased levels and activityof GSTP1 protects cells from assaults of endogenous and exogenous toxin,thereby treat and prevent benign prostatic hyperplasia, prostatitis,prevent pathogenesis of the prostate. In addition, partialisothiocyanates, such as PEITC effectively repress expressions of theandrogen receptor (AR), transcription factor Sp1, an upstream gene ofAR, and prostate specific antigen (PSA), a downstream gene of the AR toinhibit the pathogenesis of benign prostatic hyperplasic and prostatecancer. Moreover, it is worth to note that APIs in this patent areorally effective, thus they are convenient to be administered, andeasier to compliance. This characteristic is of a great advantage ofpatients with chronic prostate diseases for which long-term therapy isneeded. In this invention, we also provided evidence that PEITC waseffective against skin diseases, including, but not limited to skincancer. We showed that PEITC effectively inhibited growth of B16 mousemelanoma cells by using both in vitro and in vivo models. Our dataindicated that PEITC has wide anticancer spectrum. Many scientists havedemonstrated that PEITC is an effective agent for the chemoprevention ofvarious cancers, and now in this invention, we further demonstrated theefficacy of PEITC for the treatment and prevention of various prostatediseases. Obviously, our invention creates important transnationalmedical applications of isothiocyanates.

In order to transform the pharmaceutical active isothiocyanates intovaluable products, extensive study has been made on compositions andmanufacturing process. Compositions of pharmaceutical products anddietary supplements using isothiocynates as API are included in thispatent. Most of those compositions have been shown to have idealdissolution rate and stability. Since the effective dose of API is low,thus, we can expect that the pharmaceutical products or dietarysupplements, not only have low side effects, but the cost of theproduction will be also low, which will reduce financial burden ofconsumers. The manufacture procedures of the API have been developed inthis invention. We provided the processes to extract the API fromvegetables, or to synthesize them. As described above, this inventionprovided a novel, applicable and prospective product.

APPLICATION POSSIBILITY IN INDUSTRY

Considering the poor aqueous solubility of majority of isothiocyanatesor their derivatives, including PEITC, we provide various applicableformulations by using pharmaceutical excipients to disperse the APIfirst, thus enhance their bioavailability based on the needs ofdifferent administration. Those formulations produced ideal therapeuticeffects at low API doses and reduced side effects. Pharmacodynamicstudies using various animal models showed that the efficacy of thoseformulations were equal, might in some way better than, to Proscar orGENURIN respectively. It was a breakthrough to such chronic diseaseswhich need long term drug therapy as BPH and prostatitis. The animalstudies in this invention proved that isothiocyanates, especially PEITC,were effective against BPH and non-bacterial prostatitis at a lowdosage. Besides, the in vitro and in vivo studies also showed thatisothiocyanates, especially PEITC, which had inhibitory effect on skincancer, can be used as the drug for the prevention and treatment of theskin cancer. This invention further demonstrated that variousisothiocyanates and their derivatives can induce Phase II detoxificationenzyme, i.e. glutathione S-transferase (GSTP1); isothiocyanates, theirderivatives, and metabolites (except 4-sulfophenylisothiocyanate(SPITC), its derivatives, and metabolites) are able to inhibitexpressions of androgen receptor (AR), AR upstream gene Sp1, and ARdownstream gene, prostate specific antigen (PSA) in the human prostatecancer cell line LNCaP by molecular biological methods, In this way, thepatent provides the molecule mechanism of inhibiting benign prostateshyperplasia and prostatitis by isothiocyanates, their derivatives andmetabolites. Therefore from this invention it can be expected thatisothiocyanates, especially PEITC, and their derivatives have wideapplication possibility which may be used as pharmaceutical products,dietary supplements and cosmetic products for the treatment and/orprevention of prostatic diseases and skin cancers.

The invention claimed is:
 1. A method for the treatment of prostatitisin a subject, comprising: administrating, to the subject, a compositioncomprising an effective amount of at least one selected from the groupconsisting of an isothiocyanate compound, a derivative of anisothiocyanate compound, and a metabolite of an isothiocyanate compound,wherein the isothiocyanate compound is at least one selected from thegroup consisting of: benzyl isothiocyanate (BITC) isothiocyanate(PEITC), allyl isothiocyanate (AITC) and 4-sulfophenylisothiocyanatewhich have the following formulas (1), (2), (3), and (4), respectively;

and wherein each of said derivative and metabolite is at least oneselected from the group consisting of a glutathione-conjugate, acysteinyl glycine-conjugate, a cysteinyl-conjugate, and anN-acetylcysteine-conjugate.
 2. The method of claim 1, wherein theeffective amount is an amount effective for inducing the expression ofglutathione S-transferase (GSTP1) gene in prostate cells.
 3. The methodof claim 1, wherein the effective amount is an amount effective forinhibiting the expressions of (i) androgen receptor (AR), (ii)transcription factor Sp1, the upstream gene of androgen receptor (AR),and (iii) prostate specific antigen (PSA), the downstream gene of AR. 4.The method of claim 1, wherein each of the derivative and the metaboliteis at least one selected from the group consisting of: N-acetylcysteineconjugate of benzyl isothiocyanate (BITC-NAC), N-acetylcysteineconjugate of phenethyl isothiocyanate (PEITC-NAC), N-acetylcysteineconjugate of allyl isothiocyanate (AITC-NAC), and N-acetylcysteineconjugate of 4-sulfophenylisothiocyanate (SPITC-NAC), which have thefollowing formulas (5), (6), (7), and (8) respectively:


5. The method of claim 1, wherein the isothiocyanate compound, thederivative, or the metabolite is used alone or used as a formulationwith excipients.
 6. The method of claim 5, wherein the formulation is atleast one selected from the group consisting of a pharmaceuticalproduct, dietary supplement, food, and cosmetic product.
 7. The methodof claim 1, wherein the composition contains the following ingredients:(a) an active pharmaceutical ingredient(API), wherein said API is atleast one selected from the group consisting of an isothiocyanatecompound, a derivative of an isothiocyanate compound, and a metaboliteof an isothiocyanate compound, wherein the isothiocyanate compound is atleast one selected from the group consisting of: benzyl isothiocyanate(BITC), phenethyl isothiocyanate (PEITC), allyl isothiocyanate (AITC),and 4-sulfophenylisothiocyanate (SPITC), which have the followingformulas (1), (2), (3), and (4) respectively;

and wherein said derivative and metabolite is at least one selected fromthe group consisting of a glutathione-conjugate, a cysteinylglycine-conjugate, a cysteinyl-conjugate, and anN-acetylcysteine-conjugate, and (b) a pharmaceutically acceptablecarrier for the API, wherein said carrier is at least one selected fromthe group consisting of a surfactant, a solubilizing agent, an oilingredient, and an antioxidant; wherein the surfactant is at least oneselected from the group consisting of polyoxyethylene lauryl ether,polyoxyethylene glycol monostearate, vitamin E polyethylene glycolsuccinate, polyoxyethylene castor oil, polyoxyethylene hydrogenatedcastor oil, poloxamer, and a polysorbate; wherein the solubilizing agentis at least one selected from the group consisting ofpolyvinylpyrrolidone K17, K25, K30, or K90; and polyethylene glycol 400,4000, or 6000; wherein the oil ingredient is at least one selected fromthe group consisting of fatty acids, triglyceride, monoglyceride,diglyceride, soybean oil, corn oil, peanut oil, stearic acid, palmiticacid, palm oil, sunflower oil, olive oil, coconut oil, sesame oil,cottonseed oil, canola oil, oleic acid, linoleic acid, medium-chaintriglycerides, glyceryl monooctadecanoate, glyceryl monoacetate,glyceryl diacetate, and glyceryl triacetate; and wherein the antioxidantis a water-soluble antioxidant or a fat-soluble antioxidant, and is atleast one selected from the group consisting of vitamin C, vitamin Cpalmitate, propyl gallate, tocopherol, tert-butylated-p-hydroxyanisole,and 2,6-di-tert-butyl-p-methylphenol.
 8. The method of claim 7, whereinthe composition is at least one selected from the group consisting of atablet, a capsule, a pill, a powder for injection, an injection, alyophilized powder, an ointment, a suppository, a cream, a film, anemulsion, a spray, and an implant.
 9. The method of claim 7, wherein thecomposition is a pharmaceutical product or a dietary supplement.
 10. Themethod of claim 7, wherein the composition is administered orally,intravenously, muscularly, subcutaneously, intracavitaryly,sublingually, anally, or topically.
 11. The method of claim 1, whereinthe composition is administrated alone or in a combined therapy.
 12. Themethod of claim 11, wherein the combined therapy is at least one therapyselected from the group consisting of surgery, radiation, gene therapy,and biologics.